WO2011142832A2

WO2011142832A2 - Stem cells derived under low oxygen conditions

Info

Publication number: WO2011142832A2
Application number: PCT/US2011/000850
Authority: WO
Inventors: Maisam Mitalipova; Rudolf Jaenisch
Original assignee: Whitehead Institute For Biomedical Research
Priority date: 2010-05-13
Filing date: 2011-05-13
Publication date: 2011-11-17
Also published as: WO2011142832A3

Abstract

The invention provides human ES cell lines derived under physiological oxygen conditions. In some embodiments the invention provides female hES cell lines derived under physiological oxygen conditions, wherein the lines have two active X chromosomes and can undergo X chromosome inactivation. Methods of deriving and using the cell lines are also provided.

Description

STEM CELLS DERIVED UNDER LOW OXYGEN CONDITIONS

RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. provisional application serial number 61/334,473, filed May 13, 2010, the entire content of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Undifferentiated cells or cell clusters can be influenced by their environment. In turn, the properties or fate of the undifferentiated cell may be affected. Some environmental impacts may detrimentally affect the use of such cells. For example, variation in the environment may cause experiment to experiment variability that can be detrimental to research conducted on undifferentiated cells. The art is in need of new systems and methods for managing the impact of the environment on undifferentiated cells.

SUMMARY OF THE INVENTION

[0003] In some aspects, the present invention relates to methods and compositions for generating, maintaining, and/or culturing cells under low oxygen conditions or simulated low oxygen conditions. Certain methods and compositions of the invention find use, for example, in the preparation and maintenance of undifferentiated cells including, but not limited to, stem cells (e.g., embryonic stem cells (e.g., human embryonic stem cells), induced

pluripotent stem cells, etc.) and embryos.

[0004] In one aspect, the invention provides mammalian stem cell lines derived under physiological oxygen conditions. In some embodiments a stem cell line of the invention is a pluripotent embryonic stem (ES) cell line which, in some embodiments, is a primate ES cell line, e.g., human ES cell line. In some embodiments, the physiological oxygen conditions comprise an oxygen concentration of between about 1.5% and about 8.7%, e.g., about 5%. In some embodiments, the conditions further comprise a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%. In some embodiments a stem cell line of the invention, e.g., a pluripotent human ES cell line, has a normal karyotype. In some embodiments the pluripotent human ES cell line proliferates in culture and maintains pluripotency for at least one year. In some embodiments the cell line proliferates in culture in a genetically stable manner for at least one year.

[0005] In some aspects, the invention provides a pluripotent primate ES cell line, e.g., a human ES cell line, wherein the cell line (a) contains two X chromosomes; (b) is negative for XIST; and (c) has the ability to activate XIST expression upon differentiation. In some embodiments, both X chromosomes of said ES cell line are active. In some embodiments, the cell line exhibits biallelic expression of at least 50% of informative X-linked single nucleotide polymorphisms (SNPs). In some embodiments, the cell line is characterized in that one of said two X chromosomes becomes randomly inactivated upon differentiation. In some embodiments, the cell line proliferates in culture in a genetically stable manner, remains negative for XIST, and retains the ability to activate XIST expression upon differentiation, for at least one year. The invention further provides cell, colony, or subclone of a stem cell line of the invention, e.g., a cell, colony, or subclone of a pluripotent primate ES cell line, e.g., human ES cell line, of the invention. In some embodiments, a subclone consists of cells descended from a single cell of the cell line.

[0006] In another aspect, the invention provides non-pluripotent cells descended from a stem cell line of the invention, e.g., non-pluripotent cells descended from a pluripotent human ES cell line of the invention. In some embodiments, a non-pluripotent cell is a multipotent cell. In some embodiments, a non-pluripotent cell is obtained under physiological 02 conditions. In other aspects, the invention provides a cell culture comprising non-pluripotent descendants of a pluripotent cell line of the invention. In some embodiments, cells of the cell culture contain two X chromosomes, and, in some embodiments, the cell culture exhibits biallelic expression of X-linked genes. In some embodiments, the cells are differentiated to a desired cell lineage or type. In some embodiments, the cell culture is substantially free of pluripotent cells.

[0007] In another aspect, the invention provides a composition comprising isolated pluripotent primate ES cells, e.g., human ES cells, that were derived under physiological oxygen conditions. In some embodiments, the pluripotent cells (a) contain two X

chromosomes; (b) are negative for XIST; and (c) have the ability to activate XIST expression upon differentiation. In some embodiments, the physiological oxygen conditions comprise an oxygen concentration of between about 1.5% and about 8.7%, e.g., about 5%. In some embodiments, the conditions further comprise a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%. In some embodiments, the composition further comprises a culture medium suitable for culturing human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions. In some embodiments, the medium comprises at least one antioxidant. In some embodiments, the at least one antioxidant is selected from the group consisting of: vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

[0008] In another aspect, the invention provides a method of deriving a cell line comprising steps of: (a) obtaining an isolated early stage human embryo; and (b) deriving a pluripotent human ES cell line from the isolated early stage human embryo under

physiological oxygen conditions. In some embodiments, the isolated early stage human embryo of step (a) is a blastocyst. In some embodiments, the method comprises steps of: (i) obtaining an isolated human embryo at or before the 8 cell stage; (ii) allowing the isolated human embryo to develop to a blastocyst in culture under physiological oxygen conditions; and (iii) deriving a pluripotent human ES cell from the blastocyst under physiological oxygen conditions. In some embodiments, the method comprises: (a) isolating cells from the inner cell mass (ICM) of the blastocyst; (b) plating the ICM cells under suitable conditions such that ICM-derived outgrowths develop; (c) dispersing the ICM-derived outgrowths to obtain dispersed cells; (d) replating the dispersed cells under conditions suitable for development of hES cell colonies; and (e) culturing cells of one or more of the colonies to obtain a pluripotent human ES cell line, wherein steps (a)-(e) are performed under physiological oxygen conditions. In some embodiments, suitable conditions of step (b) comprise culturing the cells on a feeder cell layer, e.g., an embryonic feeder cell layer. In some embodiments, the cells are passaged mechanically without use of enzymatic dissociation. In some embodiments, the physiological oxygen conditions comprise an oxygen concentration of between about 1.5% and about 8.7%, e.g., about 5%. In some embodiments, the conditions comprise a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%. In some embodiments, the early stage embryo has two X chromosomes. In some embodiments, the method further comprises culturing the derived ES cell line in medium suitable for culturing human ES cells for at least two passages, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions. [0009] In some aspects, the invention provides a method of deriving a cell line comprising steps of: (a) obtaining an isolated early stage human embryo; and (b) deriving a pluripotent human ES cell line from the isolated early stage human embryo under conditions that mimic the effect of physiological 02 conditions. In some embodiments, conditions that mimic the effect of physiological 02 conditions are sufficient to preserve the XaXa status of XaXa ESCs derived under physiological 02 conditions following shift to atmospheric 02 conditions. In some embodiments, the isolated early stage human embryo of step (a) is a blastocyst. In some embodiments, the method comprises steps of: (i) obtaining an isolated human embryo at or before the 8 cell stage; (ii) allowing the isolated human embryo to develop to a blastocyst in culture under conditions that mimic the effect of physiological 02 conditions; and (iii) deriving a pluripotent human ES cell from the blastocyst under conditions that mimic the effect of physiological 02 conditions. In some embodiments, the method comprises: (a) isolating cells from the inner cell mass (ICM) of the blastocyst; (b) plating the ICM cells under suitable conditions such that ICM-derived outgrowths develop; (c) dispersing the ICM-derived outgrowths to obtain dispersed cells; (d) replating the dispersed cells under conditions suitable for development of hES cell colonies; and (e) culturing cells of one or more of the colonies to obtain a pluripotent human ES cell line, wherein steps (a)-(e) are performed under conditions that mimic physiological 02 conditions. In some embodiments, suitable conditions of step (b) comprise culturing the cells on a feeder cell layer, e.g., an embryonic feeder cell layer. In some embodiments, the cells are passaged mechanically without use of enzymatic dissociation. In some embodiments, the conditions that mimic physiological 02 conditions comprise culturing in medium comprising at least one compound that mimics the effect of physiological 02 conditions. In some embodiments, the conditions that mimic physiological 02 conditions comprise culture in medium

comprising at least one antioxidant. In some embodiments, the at least one antioxidant is selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof. In some embodiments, the early stage embryo has two X

chromosomes. In some embodiments, the method further comprises culturing the derived cell line in medium suitable for culturing a human ES cell, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

[0010] ' In another aspect, the invention provides a method of producing a population of human ES cells comprising steps of: (a) providing pluripotent human ES cells derived under physiological oxygen conditions; and (b) culturing the pluripotent human ES cells in medium suitable for culturing pluripotent human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions. In some embodiments, the culture medium comprises at least one antioxidant. In some embodiments, the at least one antioxidant is selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof. In some embodiments, the pluripotent human ES cells of step (b) are cultured under standard oxygen conditions. In some embodiments, the pluripotent human ES cells of step (a) have two X chromosomes and are negative for XIST expression, and wherein the population of step (b) remains negative for XIST expression for at least 10 passages when cultured in said medium. In some embodiments, the pluripotent human ES cells of step (a) have two active X chromosomes, and the population of step (b) retains two active X chromosomes for at least 10 passages when cultured in said medium. In some aspects, culturing ESCs derived under physiological 02 preserves at least one phenotypic characteristic of ESCs derived under physiological 02 conditions.

[0011] In another aspect, the invention provides a cell culture medium suitable for deriving or culturing human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions. In some embodiments, the compound is present in an amount sufficient to preserve the XaXa status of hESC derived under physiological 02 conditions following shift to atmospheric 02 conditions. In some embodiments, the compound comprises at least one antioxidant. In some embodiments, the at least one antioxidant is selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

[0012] In another aspect, the invention provides a kit comprising: ingredients for a cell culture medium suitable for deriving or culturing human pluriopotent cells, wherein the ingredients include at least one compound in an amount sufficient to mimic the effect of physiological 02 conditions, and wherein the kit optionally comprises at least one item selected from the group consisting of: (i) instructions for preparing the medium; (ii) instructions for deriving or culturing human pluripotent cells; (iii) serum replacement; (iv) albumin; (v) at least one protein or small molecule useful for deriving or culturing human ES cells, wherein the protein or small molecule activates or inhibits a signal transduction pathway; (vi) at least one reagent useful for characterizing human pluripotent cells; and (vii) pluripotent human cells, which are optionally ESCs of the invention. In some embodiments, the at least one compound that mimics the effect of physiological 02 conditions is an antioxidant. In some embodiments, the antioxidant is selected from the group consisting of: vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof. In some embodiments, at least some of the ingredients are dissolved in liquid. In some embodiments, at least some of the ingredients are provided in dry form. Certain conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, etc., which are within the skill of the art, may be of use in certain aspects of the invention. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in

Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., editions as of 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001 ; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Burns, R., Immunochemical Protocols (Methods in Molecular Biology) Humana Press; 3rd ed., 2005; Freshney, R.I., "Culture of Animal Cells, A Manual of Basic Technique", 5^th ed., John Wiley & Sons, Hoboken, NJ, 2005.

[0013] All patents, patent applications, publications, references, etc., cited in the instant patent application are incorporated by reference in their entirety. In the event of a conflict or inconsistency with the specification, the specification shall control. Applicants reserve the right to amend the specification based on any of the incorporated references and/or to correct obvious errors. None of the content of the incorporated references shall limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Effects of Atmospheric Oxygen Exposure on Gene Expression in hES Cells.

[0015] (A) Changes in global gene expression and hypoxia-related gene expression after acute (72hr) and chronic exposure to atmospheric (20%) oxygen. Plots represent average changes across three hES cell lines, WIBR1, WIBR2, and WIBR3. (B) Hierarchical clustering of hES cell lines maintained in 5% O₂, 20% O₂, or 5% followed by 72hr acute exposure to 20% O₂ (20A). All edges have AU p-value= 100%, demonstrating that the clusters are strongly supported by the data (Shown on the branches are the pearson correlations). (C) Flow cytometric analysis for expression of intracellular OCT4 and extracellular SSE4 in hES cells cultured either in 5% O₂, 20% O₂ (chronic), or in 5% followed by 72 hr exposure to 20% O₂ (acute), plotted as percent of positive human cells within the gates shown on right, after gating out GFP positive MEF feeders. Each data point is represented as the average of 3 experiments +/- standard deviation. Representative histograms in right panels represent cell distribution in the absence of primary antibody (control) or in the presence of primary and secondary antibodies (+1 ° Ab) for the designated antibodies.

[0016] Figure 2. X Chromosome Inactivation in hES Cells.

[0017] (A) Epifluorescence analysis of WIBR3 hES cells and their differentiated counterparts for XIST RNA (red), CotI nuclear RNA (green), and DNA (blue) in chronic 5% and 20% O₂ cultures. (B) qRT-PCR analysis of XIST transcript levels detecting two distinct exon-exon boundaries in hES cells maintained in 5% or 20% O₂ at low (<20), medium (20- 40), and high (>50) passages. Also shown are XIST expression levels after hES

differentiation in 5% (5%-Diff) and 20% (20%-Diff) oxygen as well as after 72 hour exposure to 20% O₂ after chronic culture at 5% O₂ (5-20% Acute). (C-D) Sequenom mass- spec based methylation analysis of CpG islands within the ATS promoter/enhancer region in the male hESC line WIBR1 (C) and the female line WIBR3 (D). 5% O₂ cultures are in blue, 20% O₂ in red. The location of the amplicons is plotted at the bottom of each graph with the gene annotation. The individual CpG residues are plotted relative to the XIST transcriptional start site. (E) Bisulfite sequencing analysis of six CpG sites in single clones the second (from the left) XIST promoter amplicon represented in (C-D). (F) Epifluorescence analysis of WIBR3 hES cells for XIST RNA (red), CotI nuclear RNA (green), and DNA (blue) in cultures switched either from 5% to 20% O₂ or 20% to 5% O₂ for two weeks.

[0018] Figure 3. Allele-specific Gene Expression in WIBR3 hESCs.

[0019] (A) SNP-Chip Analysis performed with all informative X-linked SNPs identified in WIBR3 hESCs. Genes are listed in order of their location relative to the pseudoautosomal region of the X-chromosome (top of the list). Genes listed are those in closest proximity to identified SNPs, with some genes represented by several SNPs listed more than once.

Monoallelic gene expression is denoted in green while biallelic expression is denoted in yellow. (B) Schematic of the X-chromosome displaying genes containing informative SNPs in line WIBR3 used for allele-specific expression analyses. The pseudoautosomal region (PAR) knovm to escape X inactivation is shown in red. (C-F) Allele-specific gene expression analysis utilizing SNPs within transcribed regions of X-linked genes (SNPs may lie within exons or introns) for Sequenom-based dye termination assays in WIBR3 ° ESCs (B), WIBR3^20% ESCs (C), WIBR3^5% ESCs after in vitro differentiation (D), or WIBR3^5% ESCs after two weeks exposure to atmospheric oxygen (E). Genes are plotted based on their location along the X-chromosome.

[0020] Figure 4. XCI in Response to Cellular Stress and Inhibition of XCI with Antioxidants.

[0021] (A) Epifluorescence analysis of WIBR2 hES cells maintained in 5% O₂ before and after undergoing stress from a freeze-thaw cycle. XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue). (B) qRT-PCR analysis of XIST transcript levels detecting two distinct exon-exon boundaries in hES cells maintained in 5% or 20% O₂, and after freeze- thaw induced stress at 5% oxygen. (C) Sequenom mass-spec based methylation analysis of CpG islands in the XIST promoter/enhancer region in the female line WIBR2 after undergoing freeze-thaw associated stress. XaXa 5% O₂ cultures are in blue, 5% O₂ stressed XaXi cultures in yellow. The location of the CpG residues is plotted relative to the XIST transcriptional start site at the bottom of each graph. (D) qRT-PCR analysis of XIST gene expression in WIBR2 hES cells cultured under 5% oxygen in the absence (control) or presence of compounds inducing cellular stress (see Table S3): HSP90 inhibitor 17AAG (24hr acute treatment), proteosome inhibitor MG132 (24hr acute treatment), organic peroxide tert-butyl hydroperoxide (t-BOOH, lOuM acute treatment and 0.5uM chronic treatment), and the γ-glutamylcysteine synthetase inhibitor L-Buthionine-sulfoximine (L-BS, 150uM acute treatment or 15uM chronic treatment) at the denoted concentrations. 24 days after introduction of compounds undifferentiated hES cell colonies were harvested for RNA analysis. XIST transcript level is normalized to GAPDH and plotted as percent expression relative to 20% oxgyen. (E) qRT-PCR analysis of XIST gene expression (using primers spanning the exon 5-6 junction) in undifferentiated, mechanically isolated hESC colonies from line WIBR2 24 days after shifting cultures from 5% to 20% oxygen, during which time the indicated antioxidant compounds were added to the culture media daily. 5% control represents hES cells not shifted to 20% oxygen, and 20% control represents untreated hES cells shifted from 5% to 20% oxygen. XIST transcript level is normalized to GAPDH and expression in 20% oxygen cultures set to 100.(F) Epi fluorescence FISH analysis of representative cultures analyzed in Figure 4A. XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue).

[0022] Figure 5. Localization of Activating and Repressive Histone Modifications.

[0023] (A) ChlP-seq localization of H3 4me3 (blue) and H3K27me3 (green) chromatin modifications along chromosome 1 in the male hESC line WIBRl and the female hESC lines WIBR2 and WIBR3. Chromosome map is shown above graphs in red box; Genome scale is indicated above graphs. (B) ChlP-seq localization of H3K4me3 (blue) and H3K27me3 (green) chromatin modifications along the X chromosome in the male hESC line WIBRl and the female hESC lines WIBR2 and WIBR3. Chromosome map is shown above graphs in red box; Genome scale is indicated above graphs.

[0024] Figure 6. Model of XCI During Development and ES Cell Culture.

[0025] Preimplantation mouse embryos exhibit imprinted silencing of the paternal chromosome (pXi) after the two cell stage which becomes reactivated in the ICM of the late blastocyst resulting in cells with two active X chromosomes (pXa mXa). Derivation of mouse ES cells results in cultures with two active X chromosomes that randomly silence one X chromosome upon differentiation. In contrast, human preimplantation embryos contain cells both with and without XIST clouds. Derivation of XaXa hESCs from human blastocysts under physiological oxygen concentration suggests the existence of cells with two active X chromosomes in late stage human blastocysts as in the mouse. Derivation of hESCs under atmospheric oxygen concentrations results in random XCI after which long periods of in vitro culture result in clonal selection and lead to monoclonal cultures expressing X-linked genes from a single X chromosome. hESC cultures maintained at physiological oxygen concentrations stably express both X chromosomes until encountering cellular stress or differentiation signals which result in random inactivation of one X chromosome. X¹* in human preimplantation embryos indicates that although an XIST cloud is present at this stage, these cells have not been formally shown to have undergone silencing of the X chromosome.

[0026] Supplementary Figure 1. Characterization of hES Cell Lines in Physiological vs. Atmospheric Oxygen Concentrations.

[0027] (A) Phase contrast micrographs of human blastocysts in culture prior to derivation (left panels), the resulting ICM outgrowths (middle panels), and colonies of established cell lines (right panels). (B) Immunofluorescence staining of hES colonies for extracellular surface pluripotency markers SSEA4 and TRA1-60 and master pluripotency regulatory network transcription factors OCT4, SOX2, and NANOG.

[0028] Supplementary Figure 2. Karyotype Analysis of 3 Pairs of hES Cell Lines.

[0029] Karyotype analyses of hES cell lines WIBR1 , 2, and 3 after chronic culture in physiological (5%) or atmospheric (20%) oxygen concentrations. Passage number is indicated.

[0030] Supplementary Figure 3. Teratoma Formation Assays.

[0031] Teratoma formation analysis from three pairs of hES cell lines depicted in

Supplementary Figure 1 showing elements from all three germ lineages: ectoderm-derived neural epithelium, endoderm-derived glandular/intestinal epithelium, and mesoderm-derived mesenchymal condensations, cartilage, and fibrous tissue.

[0032] Supplementary Figure 4. Lineage Specific Gene Expression in hES Cell- Derived Embryoid Bodies.

[0033] Quantitative RT-PCR analysis of the transcriptional activation of genes related to lineage commitment after 10 days of differentiation of hES cell lines WIBR1 , 2, 3, and the cryopreserved blastocyst-derived WIBR4 into embryoid bodies.

[0034] Supplementary Figure 5. Effects of Atmospheric Oxygen Exposure on Gene Expression in hES Cells.

[0035] (A) Heat map of 198 genes whose expression (on average across WIBR1 , WIBR2, and WIBR3) is changed in response to acute culture in 20% O₂ and which subsequently maintained that change in gene expression after chronic exposure to 20% O₂. (B) Gene set enrichment analysis for gene sets enriched in atmospheric oxygen in cell lines acutely exposed to 20% O₂, including gene sets associated with mRNA processing (p=.081 , FDR q=.238), mitochondrial activity (p=.062, FDR q=.172), and genes that are downregulated in response to hypoxia (p=.052, FDR q=.163). FDR values were derived using the C2 curated all gene set matrix. (C) Gene set enrichment analysis for gene sets enriched at 5% O₂ in comparison to cultures acutely exposed to hyperoxia (p=.018, FDR q=.442) as well as gene sets enriched at 5% O₂ in comparison to cells chronically exposed to 20% O₂ including gene sets related to hypoxia (p=.026, FDR q=.188) and gene sets related to glycolysis and gluconeogenesis (p=.008, FDR q=.218). FDR values were derived using the C2 curated all gene set matrix. (D) Changes in the expression of genes related to maintenance of pluripotency and early lineage specification plotted as fold change in chronic 20% O₂ cultures relative to 5% O₂.

[0036] Supplementary Figure 6. X Chromosome Inactivation in WIBR2 hESCs.

[0037] (A) Epifluorescence analysis of WIBR2 hES cells and their differentiated counterparts for XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue) in chronic 5% and 20% O₂ cultures. (B) qRT-PCR analysis of XIST transcript levels detecting two distinct exon-exon boundaries in hES cells maintained in 5% or 20% O₂, after hES differentiation in 5% (5%-Diff) and 20% (20%-Diff) O₂ as well as after 72 hour exposure to 20% O₂ after chronic culture at 5% O₂ (5-20% Acute). (C) Sequenom mass-spec based methylation analysis of CpG islands in the XIST promoter/enhancer region in the female line WIBR2. 5% O₂ cultures are in blue, 20% O₂ in red. The location of the CpG residues is plotted relative to the XIST transcriptional start site at the bottom of each graph. (D) Epifluorescence analysis of WIBR2 hES cells for XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue) in cultures switched either from 5%-20% O₂ or 20%-5% O₂ for two weeks. (E) Epifluorescence analysis of WIBR2^20% hES cells for XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue) at passage 1 1.

[0038] Supplementary Figure 7. FISH Analysis of X-linked Gene Expression.

[0039] WIBR3 cells cultured at 20% (top panel) or 5% (lower panel) oxygen were stained with probes against the X-linked VBP1 gene (red) and DNA (DAPI, blue).

[0040] Supplementary Figure 8. X Chromosome Paint in hES Cells at 5% and 20%

Oxygen.

[0041] WIBR3 hES cells maintained under 5% (top panels) or 20% (lower panels) oxygen were stained with X-chromosome paint (red), Cotl RNA (green), and XIST RNA (blue). Colocalization of the XIST territory with the X-chromosome domain appears as pink in the merged image.

[0042] Supplementary Figure 9. Allele-Specific Gene Expression in WIBR2 hESCs.

[0043] (A) SNP-Chip Analysis performed with all informative X-linked SNPs identified in WIBR2 hESCs. Genes are listed in order of their location relative to the pseudoautosomal region of the X-chromosome (top of the list). Genes listed are those in closest proximity to identified SNPs, with some genes represented by several SNPs listed more than once.

Monoallelic gene expression is denoted in green while biallelic expression is denoted in yellow. (B-C) Allele-specific gene expression analysis utilizing SNPs within transcribed X- linked genes (SNPs may be lie within exons or introns) for Sequenom-based dye termination assays in WIBR2^5% ESCs (B) and WIBR2^20% ESCs (C). Genes are plotted based on their location along the X-chromosome. (D) Schematic of the X-chromosome displaying genes containing informative SNPs in line WIBR2 used for allele-specific expression analyses. The pseudoautosomal region (PAR) known to escape X inactivation is shown in red.

[0044] Supplementary Figure 10. H3K4me3 and H3K27me3 Modifications Across Promoters of Known Genes in 5% and 20% Oxygen.

[0045] ChlP-seq density heat map of H3K4me3 (blue, top) and H3K27me3 (green, bottom) chromatin modifications for all annotated human genes (-20,000). The genomic region from -4kb to +4kb relative to the transcription start site of -20,000 genes is shown for each cell line and 02 condition. Gene order for each modification is determined by highest average read density in the 5% 02 condition for WIBR1 , 2, & 3, and arranged from highest to lowest density. An arrow indicates the start site and direction of transcription of the genes.

[0046] Supplementary Figure 11. Pluripotency Gene Expression in hES Cell Lines Derived from Cryopreserved Blastocysts.

[0047] Immunofluorescence staining of hES colonies for master pluripotency regulatory network transcription factors OCT4, SOX2, and NANOG and extracellular surface pluripotency markers SSEA4 and TRA1 -60 in hESC lines WIBR4, WIBR5, and WIBR6.

[0048] Supplementary Figure 12. Karyotype Analysis of hES Cell Lines Derived from Cryopreserved Blastocysts.

[0049] Karyotype analyses of hES cell lines WIBR4, 5, and 6 after chronic culture in physiological (5%) or atmospheric (20%) oxygen concentrations. The male line WIBR6 exhibited trisomy of chromosome 17 (arrow).

[0050] Supplementary Figure 13. XCI Status in hESCs Derived from

Cryopreserved Blastocysts.

[0051] Epifluorescence analysis of female WIBR4 and WIBR5 hES cells maintained in 5% oxygen (top panels), 20% oxygen (middle panels), and after differentiation of 5% oxygen cultures (lower panels). XIST RNA (red), Cotl nuclear RNA (green), and DNA (blue).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0052] The present invention relates to methods and compositions for generating, maintaining, and/or culturing cells under low oxygen conditions or simulated low oxygen conditions. The methods and compositions find use, for example, in the preparation and maintenance of undifferentiated cells including, but not limited to, stem cells (e.g., embryonic stem cells (e.g., human embryonic stem cells), induced pluripotent stem cells, etc.) and embryos.

[0053] For example, in some embodiments, systems and methods are provided for collecting, maintaining (e.g., storing), culturing, or otherwise handling undifferentiated cells in a manner that reduces the impact of environmental oxygen on the cell(s). In some such embodiments, cells are managed in a low oxygen environment. In some embodiments, cells are managed in the presence of anti-oxidants to reduce or prevent deleterious effects of oxygen on the cells. Embodiments of the present invention provide cells generated in such environments and provide for their research, therapeutic, and clinical uses (e.g., use in vitro fertilization).

[0054] Embodiments of the present invention are illustrated herein using ES cells as a model. However, it should be understood that the invention is not limited to the use of any particular cell type or its differentiation status.

[0055] I. ES Cells Derived Under Physiological Oxygen Conditions

[0056] Overview

[0057] Embryonic stem cells (often referred to as ES cells or ESCs) are pluripotent cells with the potential to develop to cells of all three germ layers (endoderm, mesoderm, ectoderm). ESCs were first derived from the inner cell mass (ICM) of pre-implantation murine embryos. Subsequently, ESCs have been derived from pre-implantation embryos of various other mammals, including primates. The present invention relates in part to derivation of pluripotent mammalian embryonic stem (ES) cells under or substantially under physiological oxygen (O₂) conditions (sometimes referred to as "low oxygen conditions" herein). In some aspects, the invention provides pluripotent mammalian ES cells and cell lines derived under or substantially under physiological O₂ conditions. The invention further provides methods of deriving pluripotent mammalian ES cells and cell lines under or substantially under physiological O₂ conditions. In certain embodiments of interest, the ES cells are primate ES cells. In certain embodiments of particular interest, the primate ES cells are human ES cells (hESC). Although the invention is mainly discussed herein in terms of human ESCs, it should be understood that the invention also provides corresponding embodiments that relate to non-human primate ESCs. In some embodiments, the non-human primate is a New World monkey such as the marmoset or squirrel monkey. In some embodiments the non-human primate is an Old World monkey such as the rhesus macaque, cynomolgus monkey, or baboon (Old World monkeys).

[0058] One of the undesired consequences of exposure of undifferentiated cells to non- physiological oxygen levels is alteration in the chromosomes, including alteration in the epigenetic state of the chromosome, which may lead to chromosomal inactivation or otherwise impact chromosome structure, stability, or gene expression, which in turn can influence the ability of a cell or groups of cells to differentiate or developed in a desired manner. Experiments conducted during the development of some embodiments of the present invention characterized the impact of oxygen on X chromosome inactivation. It is contemplated that inactivation alteration in the chromosomes, e.g., alteration in epigenetic state, may also occur in other chromosomes. Embodiments of the present invention provide for the ability to counter this impact using low oxygen conditions and/or by employing antioxidants during the management of the undifferentiated cells. In some aspects, the invention relates to hESCs that have two active X chromosomes. In certain embodiments, the ESCs retain the ability to undergo X chromosome inactivation (XCI) upon differentiation. XCI is the transcriptional silencing of one of the two X chromosomes in female mammals. During the process of XCI a large, non-coding RNA called XIST (X inactive specific transcript) is upregulated from the X chromosome to be inactivated, coats the chromosome in cis, and triggers the onset of transcriptional silencing. The other X chromosome remains active. See, e.g., Payer, B. and Lee, J.T. "X chromosome dosage compensation: How mammals keep the balance." Annu. Rev. Genet. 42:733-772, 2008. The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency exhibited by typical murine ESCs (e.g., murine ESCs derived from permissive mouse strains such as C57BL/6 or 129) with an XX karyotype. In contrast, human ESCs derived prior to the instant invention invariably exhibit signs of X chromosome inactivation (XCI) and/or defects in regulation of XIST. Described herein is the establishment of the first XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines it is demonstrated, among other things, that (1) differentiation of hESCs induces random XCI in a manner similar to typical murine ESCs, (2) exposure to atmospheric oxygen is sufficient to induce irreversible XCI and dramatic changes throughout the transcriptome, (3) the Xa exhibits heavy

methylation of the XIST promoter region, (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi; and (5) occurrence of XCI upon shift to atmospheric 02 can be prevented by use of antioxidants. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state can be preserved in vitro through culture under physiological oxygen or conditions that simulate physiological oxygen. Thus, use of physiological 02 conditions allows the derivation of pre-X inactivation hESCs. Derivation and culture in physiological oxygen also aids in maintaining pluripotency and suppressing spontaneous differentiation of hESCs. Without wishing to be bound by theory, the results described herein suggest that derivation and maintenance of hESCs under physiological 02 conditions acts to maintain hESCs in a developmentally more immature state. XCI status can serve as a marker to monitor the epigenetic status of primate ESCs, e.g., hESCs. Results described herein suggest that culture under physiological 02 conditions acts to keep hESCs in an epigenetic state that more accurately the in vivo epigenetic state of the ICM.

[0059] Furthermore, it is shown that XaXa state of hES cells is precarious and prone to X chromosome inactivation by cellular stress, e.g., oxidative stress. XCI can be inhibited by protecting hESCs from such stress, e.g., by supplementing the culture medium with appropriate protective compounds. In some aspects, the invention relates to mammalian ES cells derived at least in part under conditions that protect cells from oxidative stress, so that XCI is inhibited. In some embodiments, derivation is performed under or substantially under physiological O₂ conditions and at least in part under conditions that protect cells from oxidative stress, so that XCI is inhibited. In some embodiments, mammalian ES cells are derived and/or maintained in medium supplemented with one or more antioxidants, thereby protecting the cells from oxidative stress.

[0060] In some aspects, the invention further relates to induced pluripotent stem (iPS) cells, e.g., human iPS cells, derived under or substantially under physiological 02 conditions and/or under conditions in which the cells are protected from cell stress, e.g., oxidative stress. As known in the art, iPS cells are somatic cells that have been "reprogrammed" to a pluripotent state. iPS cells have been shown to possess certain key features of ES cells including long-term self-renewal, expression of pluripotency-associated markers, and ability to form teratomas in immune-compromised mice. iPS cells hold considerable interest in part because isogenic iPS cells can be generated from any individual. Thus, patient-specific iPS cells can readily be generated, and immune-mediated rejection can be avoided. iPS cells can be generated by infecting somatic cells such as fibroblasts with retroviruses carrying genes encoding the transcription factors Oct4, Sox2, lf4, and, optionally, c-Myc. Other combinations of factors, such as Oct4, Sox2, Nanog, and Lin28, can also induce pluripotency. Furthermore, different Klf and Sox family members can be used instead of or in addition to Klf4 and Sox2. Introduction of an appropriate set of factors causes reactivation of the endogenous pluripotency-related genes Oct4, Sox2, and Nanog, resulting in a self-sustaining pluripotent state. A variety of techniques, e.g., involving small molecules, transient transfection, infection using non-integrating viruses, and/or protein transduction have been employed in the generation of iPS cells, e.g., to replace insertion of exogenous genes encoding one or more of the factors into the genome. Furthermore, exogenously introduced genes introduced into the genome can subsequently be removed after induction of

pluripotency, e.g., by recombinase-mediated excision. Such excision may leave only a short residual sequence comprising portions of the recombinase target site. Thus, iPS cells free of introduced genetic material or essentially free of such material (e.g., containing only a residual recombinase target site or portions thereof) can be produced. See, e.g.,

PCT/US2008/004516 (WO 2008/124133) "Reprogramming of Somatic Cells";

PCT US2009/047423 (WO 2009/152529) Programming and Reprogramming of Cells;

Lyssiotis, CA., Proc Natl Acad Sci U S A., 106(22):8912-7, 2009; Carey BW, Proc Natl Acad Sci U S A; 106(1): 157-62, 2009, Soldner F, et al., Cell;136(5):964-77, 2009, and references cited in any of the foregoing.

[0061] The reprogramming process resets many of the epigenetic changes that

accompany differentiation in somatic cells. However, similar to hESCs derived under atmospheric 02 conditions, female human iPSCs exhibit XCI. Generation of hiPSCs with two active X chromosomes and/or that exhibit reduced oxygen-induced epigenetic changes would be of considerable interest. The invention encompasses deriving human iPS cells under physiological 02 conditions, wherein the resulting iPS cells exhibit two active X chromosomes. In some embodiments, the iPS cells under XCI upon differentiation and/or shift to atmospheric 02 conditions.

[0062] In some aspects, the invention relates to pluripotent or multipotent stem cells obtained from other sources, such as amniotic stem cells (e.g, isolated from amniotic fluid), cord blood stem cells, fetal stem cells, or stem cells isolated from a juvenile or adult mammal. [0063] Cell Culture Under Physiological02 Conditions

[0064] Mammalian cell culture, including derivation of ES cells, involves maintaining cells in a culture vessel (e.g., a cell culture dish) containing an appropriate culture medium and under conditions suitable for cell survival and proliferation. In general, appropriate culture media comprise appropriate nutrients (e.g, amino acids and sugar(s)), vitamins, salts, and other components to permit survival and (usually) proliferation of one or more cell types of interest. Maintaining cells in culture includes performing manipulations such as media changes and passaging (also known as splitting or subculturing) as appropriate. Media changes involve removing at least some of the medium from a culture vessel and replacing with new medium. Passaging involves transferring at least some cells from a first culture vessel into a different culture vessel, optionally with some of the medium, and adding fresh medium. Such manipulations are usually carried out in an enclosed work space termed a "tissue culture hood" , "biosafety cabinet", or the like, typically designed to provide an aseptic environment. For purposes of this invention, a work space suitable for performing cell manipulations is referred to as a "working chamber". When such manipulations are not being performed, culture vessels are usually housed in an incubator capable of providing control over various parameters such as temperature and carbon dioxide (C02) concentration.

[0065] Mammalian cells are typically cultured under gas mixtures (e.g., in incubators and working chambers) having an 02 concentration similar to that of the earth's atmosphere, i.e., about 20% (mole fraction), and without independently controlling the 02 concentration in the culture medium or in the space between the culture medium and the culture vessel lid (termed the "headspace"). The P02 of the culture medium is determined mainly by diffusion of 02 from the gas mixture, so culture under these conditions results in medium having a P02 equal to about 142±10 mmHg assuming 37°C, >95% relative humidity, and approximately standard atmospheric pressure. For purposes of this invention, such conditions are referred to as "atmospheric 02 conditions".

[0066] Physiological O₂ conditions involve culture under O₂ concentrations and P02s that are less than half those of atmospheric conditions. Such 02 conditions are significantly closer to those present in the mammalian oviduct and uterus than are atmospheric conditions. Physiological 02 conditions may, but need not be, selected to match a value measured in vivo (i.e., in the mammalian oviduct or uterus). Thus particular 02 conditions selected within the range of physiological 02 conditions may differ from those present in the mammalian oviduct or uterus of a particular species. In some aspects, "physiological 02 conditions" refers to culture in medium having a P02 between 10 mmHg and 60 mmHg, which corresponds to gas mixtures having between about 1.5% and about 8.7% 02 concentration (assuming culture at approximately 37°C, >95% relative humidity, pressure approximately equal to standard atmospheric pressure).

[0067] Physiological 02 conditions can be achieved by using incubators and working chambers containing gas mixtures having 02 concentrations controlled to be between about 1.5% and about 8.7%, without independently controlling the 02 concentration of the culture vessel headspace or the P02 of the culture medium itself. For purposes of this invention, O₂ conditions will usually be described in terms of the 02 concentration of a gas mixture under which cells are cultured, e.g., the 02 concentration of a gas mixture in an incubator or working chamber. However, it should be understood that physiological O₂ conditions encompass conditions in which cells are cultured in media that has a P02 between 10 mmHg and 60 mmHg, wherein such PO2 is achieved by other means. For example, the 02 concentration of the gas mixture in the headspace and/or the P02 of the culture medium could be monitored and, optionally, independently controlled, e.g., by introducing or removing appropriate gases directly into or from the headspace and/or medium. In some embodiments of the invention, culturing cells under physiological 02 conditions includes using culture medium that has been at least partially degassed and/or equilibrated with a gas mixture having an 02 concentration between about 1.5% and about 8.0% for performing at least some media changes and/or passaging. In other embodiments, media that has not been degassed or equilibrated is used. For purposes of this invention, use of such media is within the scope of physiological 02 conditions.

[0068] In some embodiments of the invention, physiological 02 conditions include maintaining cells under a gas mixture having an O₂ concentration between 2.0% and 7.0%, e.g., between 4.0% and 6.0%, e.g., between 4.5% and 5.5%. Thus, physiological 02 conditions encompass use of media having P02 values corresponding to an O₂ concentration between 2.0% and 7.0%, e.g., between 4.0% and 6.0%, e.g., between 4.5% and 5.5%. For example, the P02 can be about 20-50 mmHg, e.g., about 30-40 mmHg, e.g., about 36 mmHg. Opening an incubator or working chamber may cause the concentrations of gases inside to transiently deviate from selected concentration(s). Furthermore, in some embodiments of the invention culture vessels (typically without removing the lid) may be transported between an incubator and a working chamber or examined briefly under a microscope in ambient air. Changes in the P02 of the culture medium, if any, caused by such occurrences would be minor, would not be expected to shift the P02 outside physiological 02 conditions, and are therefore encompassed within the phrase "under physiological 02 conditions".

[0069] A gas mixture or culture medium of use in the invention contains one or more other gases in addition to O₂. Such other gases may consist primarily or essentially completely of N₂ and CO₂, as in standard cell culture. Such gases are usually supplied as compressed gas cylinders. It will also be understood that other gases, such as those found in the earths's atmosphere, may be present (e.g., as impurities in 02, N2, and C02 supplies). Typically any such gas(es) would be present in small amounts, e.g., totalling less than 1 -2% of the gas molecules present. In certain embodiments the O₂, N₂, and CO₂ used in an incubator or working chamber is at least 99% pure, e.g., about 99.5% pure. For example, medical grade gases can be used. For purposes of description herein it is assumed that the O₂, N₂, and CO₂ are 100% pure. Thus the concentrations of N₂ and CO₂ may be selected so that the sum of the concentrations of O₂, N₂, and CO₂ equals 100%. The gas concentrations as described herein consider only the dry components. It will be understood that the gas mixture may also include water vapour. For example, the culture conditions, e.g., in the incubator, may include a relative humidity level of about 80%-95%, which may be achieved as known in the art. The C02 concentration used in the incubator is often significantly higher than that in ambient air in order to help control the pH of the medium. In some embodiments, the gas mixture contains at least 80% N₂, up to about 20% CO₂, and between 1.5% and 8% 02. In some embodiments, the gas mixture contains between 80% and 90% N₂ or between 90% and 95% N₂. In some embodiments, the gas mixture contains between 2% and 10% CO₂. In some embodiments, physiological O₂ conditions include about 5% O₂, about 92% N₂, and about 3% CO₂. In some embodiments, physiological O₂ conditions include 5±1% O₂, 92±1% N₂, and 3±1% CO₂, e.g., 5% O₂, 92% N₂, and 3% CO₂. It will be understood that the gas concentrations in an incubator or working chamber may fluctuate slightly over time. For example, in some embodiments O₂ and CO₂ concentrations are controlled to within ±1.0%, e.g., within ±0.5%, e.g., within ±0.2% while the incubator or working chamber is closed.

[0070] Gas concentrations, e.g., in an incubator or working chamber, can be measured and controlled using methods known in the art. 02 concentration can be measured using, e.g., zirconium oxide sensors; C02 concentration can be measured using, e.g., thermal conductivity (TC) or infrared (IR) sensors. Electrode systems can be used to measure 02 and/or C02 tensions in medium. A suitable controller, e.g., a feedback controller such as a proportional-integral-derivative (PID) controller may be used in conjunction with the sensors to regulate gas inflow so as to maintain desired gas concentrations.

[0071] In some embodiments of the invention, a cell culture incubator that offers accurate control over both 02 and C02 concentrations is used. Such incubators are available from a variety of different manufacturers, such as Thermo Fisher Scientific (Waltham, MA), Sanyo Electric Biomedical Co. (Tokyo, Japan). For example, the Heracell® 150i and 240i (Thermo Fisher Scientific) permit the user to select an 02 concentration between 1 % and 21 % or between 5% and 90% and a C02 concentration between 0% and 20%. The remainder of the gas can be N2. In some embodiments, a sealable chamber equipped with a device that provides control over 02 concentration ("02 controller") is used for maintenance and/or manipulation of the cells. For example, a chamber equipped with an 02 controller could be placed in an incubator that does not provide control over 02 concentration. Such chambers could also be used for manipulation of the cells. 02 controllers and chambers equipped with such devices are available from a variety of manufacturers such as Biospherix (Laconia, NY) For example, ProOx oxygen controllers provide means to control single-setpoint oxygen in chambers, incubators, gloveboxes and other semi-sealable enclosures. In some embodiments, the 02 controller utilizes an 02 sensor, which monitors the 02 concentration within the chamber. When the 02 concentration increases above a desired level, the controller causes infusion of N2 and/or C02 fiom a gas supply until the chamber reaches the desired 02 concentration. Optionally, a C02 controller can be used in addition, in order to control C02. HypOxygen (Frederick, MD) offers workstations with working chambers that allow control over 02 and C02 concentrations.

[0072] In some embodiments, a glove box is used for culturing, e.g., manipulating, embryos and/or cells. A glove box comprises a sealed container that allows a user to manipulate objects under conditions different from that in which the user is located. Gloves are built into the sides of the glovebox and arranged so that the user can place his or her hands into the gloves and perform tasks inside the box without breaking containment. Part or all of the box is usually transparent to allow the user to see what is being manipulated. A glove box of use in the present invention allows a user to manipulate cells, culture vessels, and other items useful in cell culture, in a gas mixture that differs from that of the air in which the user is located, e.g., a gas mixture containing between 1.5% and 8.0% 02. In some embodiments, a glove box has an opening to allow items to be placed inside, which may entail breaking containment for a short period of time. In some embodiments, items can be transferred into a glove box without breaking containment. A glove box would typically be used as a working chamber but could serve as an incubator as well.

[0073] In some embodiments, an incubator or working chamber is designed such that the 02 concentration and, optionally, the C02 concentration return to desired concentration(s) within 5-15 minutes or within 15-30 minutes following a deviation from the selected concentration(s). In some embodiments of the invention, the 02 concentration of the gas mixture in an incubator and working chamber are selected to be the same or about the same (e.g., within ±1% or within ±2%), thus reducing the variability in 02 concentration to which cells are exposed. For example, if the incubator is set to 5% 02, the working chamber may be set to 5% or to between 4% and 6%. In some embodiments, it may be desirable to transport embryos and/or cells between buildings, between different areas of a building, etc. If desired, a sealed chamber containing a gas mixture at physiological 02 concentration can be used for such transport.

[0074] In some embodiments, cells are maintained at least in part in a room that is kept at physiological 02 conditions. The 02 concentration of incubators and/or working chambers in such a room may or may not be directly controlled.

[0075] Embryo Culture and ES Cell Line Derivation

[0076] As used herein, "derive", "derivation", and similar terms refer to the process of generating an ES cell or ES cell line in vitro (i.e., outside the body). An "ES cell line" is a population of ES cells that has proliferated in vitro through at least 2 passages after the initial appearance of ES cells and retains capacity for further proliferation. For example, an ES cell line may have been or may be capable of being maintained as a proliferating culture for an extended period of time (e.g., months, years, indefinitely) with appropriate passaging. ES cells are typically derived as an ES cell line. Therefore, deriving ES cells and deriving ES cell lines are referred to interchangeably herein. In some embodiments derivation is performed under or substantially under physiological O₂ conditions. In some embodiments, derivation is performed at least in part under conditions that protect cells from oxidative stress, so that XCI is inhibited, as discussed further below. In some embodiments, derivation is performed under or substantially under physiological O₂ conditions and at least in part under conditions that protect cells from oxidative stress, so that XCI is inhibited.

[0077] Any suitable procedure for deriving mammalian ES cells may be used in various embodiments of the invention. Techniques and reagents suitable for deriving and

maintaining ES cells from various mammalian species are known in the art and are described in the literature. See Hogan, B., et al., Manipulating the Mouse Embryo, A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003 for nonlimiting descriptions of techniques for deriving murine ES cells. See e.g., U.S. Pat. No. 6,200,806 and Thompson, J. A. et al. Science, 282: 1 145-7, 1998 for nonlimiting descriptions of techniques for deriving primate ES cells. ES lines have been derived from representative New World and Old World monkey species, including at least the marmoset (a New World monkey), and the rhesus macaque, cynomolgus monkey, and baboon (Old World monkeys), as well as humans. Additional description of isolating non-human primate ESCs is found in Navara, C.S., et al., Curr Protoc Stem Cell Biol.; Chapter 1 :Unit 1A.1 , 2007. Additional description of methods and materials of use for deriving and/or culturing hESCs are found, e.g., in, Mitalipova, M. and Palmarini, G., "Isolation and Characterization of Human

Embryonic Stem Cells"in Turksen, K. (ed.) Human Embryonic Stem Cell Protocols, Methods in Molecular Biology, Vo. 331 Humana Press, Inc. Totowa, NH, 2006, the entire contents of which are incorporated herein by reference. Additional protocols are described in Ilic D, et al. Derivation of hESC from intact blastocysts.; Curr Protoc Stem Cell Biol.;Chapter l :Unit 1A.2, 2007; and Chen, AE and Melton, DA. "Derivation of Human Embryonic Stem Cells by Immunosurgery" J Vis Exp. (10): 574, 2007.

[0078] In some embodiments of the invention, rodent (e.g., rat or rabbit), ungulate (e.g., horse, cow, goat, pig, sheep), canine, or feline ES cells or ES-like cells are derived under or substantially under physiological 02 conditions. As used herein, an "ES-like" cell is a cell that has been shown to possess some characteristics of ESCs but does not meet art-accepted criteria for genuine ESCs. For example, such cells may lack capability for long-term proliferation in culture in a pluripotent state in which they are able to give rise to cells of all three germ layers using art-accepted tests such as teratoma formation and/or may fail to be capable of contributing to chimeras. Derivation of putative ESCs or ES-like cells from various non-primate mammals such as pigs and cows has been reported. See, e.g., Chen, L. , et al. Establishment of pluripotent cell lines from porcine preimplantation embryos. Theriogenology, 52, 195-212, 1999; Mitalipova, M., et al., Pluripotency of bovine embryonic cell line derived from precompacting embryos. Cloning, 3, 59-67, 2001. However, reproducible derivation and maintenance of ESCs from a number of non-primate mammalian species has been challenging. The present invention encompasses improving the derivation and/or maintenance of ESCs from a non-primate mammal (e.g., an ungulate) by performing the derivation process under physiological 02 conditions and, optionally, maintaining the resulting cells under physiological 02 conditions. In some embodiments the invention provides ESCs or ES-like cells derived under physiological 02 conditions from endangered or threatened mammalian species or commercially significant mammalian species, e.g., domesticated species, e.g., pets or farm animals. As used herein, a "commercially significant species" is one whose cultivation or sale contributes at least $ 100,000 to the gross domestic product (GDP) of at least one country and/or that is an object of interstate or inter-country commerce or tourism. As used herein, an "endangered or threatened species" is one listed as being endangered or threatened by a government agency responsible for making such listings.

[0079] ES cell lines are typically derived from an early stage embryo or a portion thereof such as a cell or group of cells isolated from the embryo. In this context, cell(s) that have been separated from at least some other cells or structures of the embryo are considered "isolated". For example, a cell is considered "isolated" if it is no longer part of an intact embryo, e.g., it has been removed from the embryo or if the embryo has at least in part disintegrated or been manipulated so that it is no longer a single cohesive structure. As used herein the term "early stage embryo" encompasses pre-implantation embryos from the first cell division up to and including the blastocyst stage. In some embodiments, an ES cell line is derived from cells obtained from a blastocyst stage embryo from which the zona pellucida (ZP) has been at least in part removed, e.g., substantially removed. In some embodiments, an ES cell line is derived from cells obtained from a blastocyst stage embryo. For example, an ES cell line is often derived from the inner cell mass (ICM) of a blastocyst. Isolation of ICM comprises removal of, or separation from, the ZP). Optionally, isolation of ICM can include removal or lysis of at least some trophectoderm cells (e.g., by mechanical removal, laser- assisted removal, and/or complement-mediated lysis). In some embodiments, an ES cell line is derived from an embryo in the morula stage or earlier. In some embodiments, an ES cell line is derived from an isolated blastomere obtained from an early stage embryo that has not reached the blastocyst stage, e.g., an isolated blastomere obtained from a 4-8 cell stage embryo.

[0080] In some embodiments, an ES cell line is derived from an early stage embryo produced using in vitro fertilization (IVF). For example, the embryo may have been created for reproductive purposes and is not needed for such purposes. Standard methods of performing IVF and culturing the resulting zygote and early stage embryo and known in the art can be used. A typical, non-limiting embodiment is described below.

[0081] When follicular maturation is judged to be adequate, human chorionic

gonadotropin (hCG) is given. This agent, which acts as an analogue of luteinising hormone, would cause ovulation about 42 hours after injection, but a retrieval procedure takes place just prior to that, in order to recover the egg cells from the ovary. The eggs are retrieved from the patient using a transvaginal technique involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be aspirated, and the follicular fluid is handed to the IVF laboratory to identify ova. It is common to remove between ten and thirty eggs.

[0082] In the laboratory, the identified eggs are stripped of surrounding cells and prepared for fertilisation. In the meantime, semen is prepared for fertilisation by removing inactive cells and seminal fluid in a process called sperm washing. If semen is being provided by a sperm donor, it will usually have been prepared for treatment before being frozen and quarantined, and it will be thawed ready for use. The sperm and the egg are incubated together at a ratio of about 75,000: 1 in the culture media for about 18 hours. In most cases, the egg will be fertilised by that time and the fertilised egg will show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using intracytoplasmic sperm injection (ICSI). The fertilised egg is passed to an appropriate growth medium and left for about 48 hours until the egg consists of six to eight cells.

[0083] Culture of embryos is performed in an artificial culture medium. Autologous endometrial coculture (on top of a layer of cells from the woman's own uterine lining) is occasionally used. With regard to artificial culture medium, the same culture medium can be used throughout the period, or a sequential system can be used, in which the embryo is sequentially placed in different media. For example, when culturing to the blastocyst stage, one medium may be used for culture to day 3, and a second medium is used for culture thereafter. Artificial embryo culture media typically contain glucose, pyruvate, and energy- providing components, but addition of amino acids, nucleotides, vitamins, and cholesterol can improve embryonic growth and development.

[0084] Laboratories have developed grading methods to judge oocyte and embryo quality. Embryos can be graded based on the number of cells, evenness of growth and degree of fragmentation.

[0085] In some embodiments of the invention, cells used for in vitro fertilization are maintained in a physiological oxygen or simulated physiological oxygen environment prior to IVF. In some embodiments, sperm and/or oocytes are stored frozen in the presence of one or more antioxidants prior to use in an IVF procedure. The cell maintenance and/or storage approaches of the present invention may be applied to any desired in vitro fertilization process. It is contemplated that improved implantation rates of viable embryos are achieved using embodiments of the present invention. For example, embryos that have reached the 6- 8 cell stage are transferred to a subject's uterus three days after retrieval. In many instances however, embryos are placed into an extended culture system with a transfer done at the blastocyst stage at around five days after retrieval, especially if many good-quality embryos are still available on day 3. Blastocyst stage transfers have been shown to result in higher pregnancy rates.

[0086] In some embodiments, fertilization is performed under physiological 02 conditions. In some embodiments, fertilization is performed under non-physiological 02 conditions, and the resulting zygote is transferred to physiological 02 conditions and maintained under physiological 02 conditions until use in ESC derivation. In some embodiments, fertilization is performed under non-physiological 02 conditions, and the early stage embryo is maintained under non-physiological O₂ conditions, e.g., atmospheric 02 conditions, for at least some time. For example, in some embodiments, the embryo is maintained in non-physiological 02 conditions up to the blastocyst stage and, optionally, including part of the blastocyst stage, until use in ESC derivation. In some embodiments the embryo is transferred to physiological 02 conditions at the blastocyst stage and, optionally, maintained under physiological 02 conditions for an additional time period until use in ESC derivation. In some embodiments, the embryo is transferred to physiological 02 conditions at or before the 4-8 cell stage and, optionally, maintained under physiological 02 conditions for an additional time period until use in ESC derivation. For example, the embryo can be maintained under physiological 02 conditions until the blastocyst stage and then use in ESC derivation. In some embodiments, the embryo is transferred to physiological 02 conditions between the 8 cell stage and the blastocyst stage and, optionally, maintained under physiological 02 conditions for an additional time period until use in ESC derivation.

[0087] In some embodiments, the early stage embryo has not been cryopreserved. In some embodiments, the embryo a poor quality embryo unsuitable for reproductive purposes within generally accepted practice. For example, the embryo may have been graded using an established grading system and determined to be of poor quality. In some embodiments pre- implantation genetic diagnosis (PIGD) has been performed and revealed that the embryo has a genetic mutation, polymorphism, chromosomal abnormality, or other indicator of a genetically or epigenetically determined disease or syndrome.

[0088] In some embodiments, a zygote or early stage embryo has been cryopreserved. Methods for cryopreserving zygotes and early stage embryos, e.g., employing vitrification or slow freezing, are known in the art. See, e.g., and references therein. In some embodiments the embryo is allowed to develop in culture until, e.g., the 4-8 cell stage, or to any stage up to and including the blastocyst stage, and then cryopreserved. In some embodiments the embryo is cultured under non-physiological O₂ conditions, e.g., atmospheric 02 conditions, prior to cryopreservation, while in other embodiments the embryo is cultured under physiological O₂ conditions prior to cryopreservation. In some embodiments, the

cryopreserved zygote or embryo is placed under physiological 02 conditions during or after thawing. In some embodiments, the zygote or embryo is placed under physiological 02 conditions immediately after thawing (e.g., within 5-10 minutes after thawing). In some embodiments, the zygote or embryo is placed under physiological 02 conditions within 1 -2 . hours, or within up to 24 hours after thawing.

[0089] In some embodiments, an ESC line is derived from an embryo produced using somatic cell nuclear transfer (SCNT). In this technique, the nucleus is removed from a normal egg, thus removing the genetic material. A donor diploid somatic cell is placed next to the enucleated egg and the two cells are fused, or the nucleus is introduced directly into the oocyte, e.g., by micromanipulation. The fused cell has the potential to develop into an embryo, which may then be used in the ES cell derivation process. Often, the donor nucleus and the recipient enucleated cell are of the same species. Parthenogenesis can also be used to produce embryos, e.g., from oocytes, from which ESCs can be derived. [0090] Derivation of an ES cell line begins when an early stage embryo or a portion thereof such as individual blastomere(s) or ICM cells, or other cellular material suitable for deriving ESCs is placed under culture conditions appropriate for deriving ES cells. In some embodiments, manipulations such as removal of the ZP, removal of trophectoderm cells, or isolation of ICM or blastomere(s) for ESC derivation is performed under physiological 02 conditions. In other embodiments, such manipulations are performed under non- physiological 02 conditions, and the embryo or cell(s) are placed under physiological 02 conditions immediately thereafter isolation (e.g., within 5-10 minutes).

[0091] Appropriate conditions for ESC derivation include placing the early stage embryo or portion thereof in a suitable culture medium at a suitable temperature (usually 37±1 °C). A variety of media suitable for deriving ESCs are known in the art. A basal media for deriving and/or culturing ESCs comprises an energy source (typically a sugar) and metabolic building blocks such as at least the essential amino acids, as well as vitamins and inorganic salts, and can contain various other components such as one or more lipid(s), sodium pyruvate, glutamine, putrescine, nucleotide(s), non-essential amino acids, buffers, and/or pH indicators. Antibiotics can be added to the media if desired. Suitable media include DMEM, mixtures of DMEM and F12 (e.g., a 1 : 1 mixture), or other media containing the same or similar components. In some embodiments a reduced osmolality medium (as compared with DMEM or DMEM/F12) is used, such as KnockOut™-DMEM or KnockOut™-DMEM/F12 (Invitrogen, Carlsbad, CA). An ESC derivation medium can comprise one or more growth factors, and/or signalling pathway activators or inhibitors. In some embodiments, the medium comprises a fibroblast growth factor (FGF), e.g., FGF2 (also called bFGF), FGF4, FGF9, FGF17 and/or FGF 18. In some embodiments, medium for deriving primate, e.g., human, ESCs, may comprise at least about 4 ng/ml bFGF, e.g., between about 4 ng/ml bFGF and about 500 ng/ml bFGF. In some embodiments, the medium comprises a STAT3 pathway activator, e.g., a leukemia inhibitory factor (LIF) receptor agonist, e.g., LIF. For example, a medium can comprise at least 1 g/ml LIF, e.g., between about 1 μg/ml and about 500 μg/ml LIF. For example, medium for deriving primate, e.g., human, ESCs, may comprise about 15 ng/ml bFGF and about 10 μg/ml leukemia inhibitory factor (LIF). In some embodiments, derivation medium comprises serum (e.g., fetal bovine serum (FBS)), while in other embodiments a serum-free medium is used. In some embodiments, the medium contains up to about 20-25% serum. An exemplary serum-containing medium comprises DMEM/F12 (1 : 1 mixture), 20%FBS, human LIF (hLIF), and human FGF2 (hFGF2). Serum-free media typically comprise components that can substitute for components present in serum ("serum replacement"). Such components may be supplied in concentrations that are greater than those used in serum-containing medium. For example, FGF2 may be supplied at a concentration greater than 4 ng/ml. In some embodiments, FGF2 is provided at a concentration of between 80 ng/ml and 200 ng/ml, e.g., about 100 ng/ml. Commercially available reagents such as Knockout™ Serum Replacement (KSR) or KnockOut™ SR XenoFree (a defined human origin, xenofree serum replacement supplement), can be used in certain embodiments (Invitrogen, Carlsbad, CA). See, e.g., WO 98/30679 for description of components in serum replacement KSR. In some embodiments the medium contains up to about 10% KSR. Albumin (e.g., bovine serum albumin or human albumin) is another component commonly found in ESC derivation medium. In some embodiments, the medium comprises Plasmanate® (Bayer Biological), a solution comprising a mixture of human plasma proteins including albumin and alpha and beta globulins. In some embodiments, a medium comprises both serum and at least some serum replacement components. For example, a medium may comprise between 1 and 10% KSR. An exemplary medium comprises 75% DMEM/F12 (1 : 1 mixture), 7% fetal bovine serum, 7% KO serum

replacement, 7% human Plasmanate, hLIF, and hFGF2. In some embodiments the derivation medium is unfavorable for proliferation of trophectoderm cells. mTeSR™ (Ludwig T, et al., Nat Biotechnol 24: 185-187, 2006 is a commercially available serum-free medium (StemCell Technologies, Vancouver, BC). See, e.g., Ludwig T, A and Thomson J. Defined, feeder-independent medium for human embryonic stem cell culture., Curr Protoc Stem Cell Biol., Chapter l :Unit 1C.2, 2007.

[0092] In some embodiments, "feeder cells" are used in the derivation process. For example, the embryo or portion thereof, e.g., ICM, can be plated on a layer of feeder cells. As known in the art, the term "feeder cells" refers to cells of a first type that are co-cultured with cells of a second type, usually in order to provide an environment that promotes the survival and/or proliferation of the latter and/or their acquisition or maintenance of a phenotype of interest. For example, the feeder cells may secrete soluble substances e.g., signaling molecules or growth factors into the medium or provide a substrate that promotes such survival, proliferation, or phenotype. Commonly used feeder cells include mouse embryo fibroblasts (MEFs) or human fibroblasts, e.g., human foreskin fibroblasts. Typically feeder cells are substantially non-proliferating, e.g., they have been "inactivated" by treatment with an inhibitor of cell division such as mitomycin C or by radiation. In some embodiments ESCs are derived under conditions such that a resulting ESC culture is free or essentially free of feeder cells. In some embodiments, an ESC culture or composition is essentially free of feeder cells if no more than 2.5% of the cells present in the culture or composition are feeder cells, e.g., no more than about 1%, 0.5%, 0.25%, 0.1%, 0.05%», or 0.01% of the cells are feeder cells. For example, in some embodiments, feeder cells are not used in the derivation process. In some embodiments, an embryo or portion thereof is initially plated on feeder cells and/or maintained on feeder cells for one or more passages and are then subjected to one or more passages without use of feeder cells, so that the resulting ESC culture is free or essentially free of feeder cells. If desired, the presence of feeder cells can be assessed and optionally quantified, using methods known in the art, such as examining the culture for expression of markers typically expressed by such cells. In some

embodiments, feeder cells are genetically modified to express a readily detectable marker such as a fluorescent protein (e.g., green fluorescent protein), thereby facilitating their detection. Feeder-free conditions can comprise (i) use of a suitable substrate, which can comprise, e.g., an extracellular matrix-like or basement membrane-like material such as Matrigel (BD Biosciences), CELLstart™ (a humanized substrate for stem cell culture, Invitrogen, Carlsbad, CA), specific proteins such as fibronectin, laminin, vitronectin, collagen(s) or mixtures thereof, or synthetic materials; and/or (ii) use of conditioned medium (e.g., MEF-conditioned medium), growth factors, and/or signalling molecules (e.g., signalling pathway activators or inhibitors) to substitute for feeder cells. As known. in the art, the term "conditioned medium" refers to cell culture medium in which cells have been previously cultured. A conditioned medium typically contains soluble substances, e.g., signaling molecules or growth factors, that are produced by the cells during their cultivation and released into the medium. Such substances can promote the survival and/or proliferation of cells that are subsequently cultured in the conditioned medium and/or promote the acquisition or maintenance of a phenotype of interest by such cells. In some embodiments a medium containing a mixture of conditioned and unconditioned ("fresh") medium is used. For example, a medium can contain between 1% and 99% conditioned medium by volume, with the remaining medium being unconditioned medium. In some embodiments, a medium contains between 5% and 75%> conditioned medium, e.g., between 10% and 50%. [0093] In some embodiments, derivation of hESC lines occurs under xeno-free conditions, i.e., the culture media, growth substrate, and other materials that contact the cells are free or essentially free of biological substances obtained from non-human animals. In some embodiments, the medium is chemically defined, e.g., free of serum and tissue/cell extracts. Optionally, protein components used in the derivation and/or maintenance of hESCs are human proteins and/or are recombinanfly produced, e.g., in human cells.

[0094] The culture is maintained under physiological 02 conditions and monitored for emergence of ES cells, which typically appear as morphologically distinctive colonies. . Media changes and passages can be performed as required. ES cells and ES cell colonies are readily recognized by those skilled in the art. For example, primate ES colonies exhibit a compact morphology (tightly packed cells) and sharp colony boundaries and contain cells with high nucleus to cytoplasm ratios and prominent nucleoli. Areas of differentiated cells can appear at the edges of ES cell colonies, especially if the cells become crowded. Such cells are often larger and flatter. If desired, such cells can be substantially removed during mechanical passaging.

[0095] In some embodiments of the invention, derivation of an ES cell line is considered to have occurred once ES cells, e.g., ES cell colonies, have appeared and been passaged at least once, wherein a "passage" event comprises collection of at least some of the ES cells in a culture vessel, transfer of at least some of the collected cells to a new vessel, and

proliferation of at least some of the transferred ES cells. In some embodiments, cells are transferred in a dispersed or mostly dispersed state as pools of cells from one or more colonies. In some embodiments, cells are transferred mechanically as larger colony fragments from an individual colony or from multiple colonies. ES cell proliferation may be evident by expansion of transferred ES cell colon(ies) or colony fragments or emergence of new ES cell colonies. In some embodiments, derivation further comprises passaging ES cells at least 1-4 more times. Optionally, the composition of the culture medium is changed during these passages. For example, the concentrations of certain components such as serum, serum replacement, growth factors, signalling pathway activators or inhibitors, etc., are increased or decreased. The ESCs may be switched from ESC derivation medium to ESC maintenance medium during these passages. For example, LIF may be eliminated.

[0096] One aspect of the invention involves dividing the cultured cells into multiple subcultures (e.g., 2, 3, 4, 5, or more individual cultures) during the derivation process and subjecting the subcultures to different culture conditions of interest during the remainder of the derivation process, and optionally thereafter. For example, the culture could be divided after the appearance of ICM outgrowth. Culture conditions of interest could vary with respect to 02 concentrations, culture media, substrates, passaging techniques, or other parameters. The resulting ESC lines can be compared, e.g., with respect to any of the characteristics described herein. Since the lines are genetically substantially identical, the confounding factor of genetic variability is avoided, thus facilitating the correlation of particular culture conditions with particular phenotypes. This approach can be used, for example, to compare the effects of different 02 concentrations (e.g., 2% 02 vs 5% 02) or to identify compounds that mimic the effects of physiological 02 conditions. The invention provides sets of ESC lines, wherein each set comprises at least 2 cell lines that are substantially genetically identical, and wherein the lines were derived under culture conditions that differ with respect to at least one parameter.

[0097] ES Cell Line Characteristics

[0098] Mammalian ESCs have the ability to differentiate into cells of the three germ layers. In addition, ES cell lines of the invention can have a number of other characteristics of interest. Characteristics of interest can include, e.g., expression level of one or more genes of interest (which can be assessed at the level of RNA or protein), karyotype, self-renewal capacity, telomere length, DNA methylation profile (e.g., within a particular DNA region), histone modification profile (e.g., within a particular DNA region), differentiation capacity, and XCi status (for cell lines having multiple X chromosomes).

[0099] Techniques for assessing characteristics of interest are known in the art. Such techniques include, e.g., visual inspection, flow cytometry, immunofluorescence, quantitative real-time reverse-transcriptase PCR, G-banding, DNA and/or RNA fluorescence in situ hybridization (FISH), Cot-1 analysis, immunocytochemistry, enzyme assays, microarray analysis, DNA sequencing (optionally using high throughput sequencing methods such as massively parallel sequencing, e.g., using the Illumina platform (Illumina, San Diego, CA)), bisulfite sequencing, chromatin immunoprecipitation and microarray analysis (ChlP-Chip), chromatin immunoprecipitation and sequencing (ChlP-Seq), allele-specific expression analysis, differentiation assays (e.g., embryoid body (EB) formation assay, teratoma formation assay). [00100] Whether a cell line (or other cell population) exhibits a particular characteristic is usually determined by assessing a representative sample of the population. The number of cells in the sample typically depends on the characteristic being assessed and the assessment technique. Certain characteristics may be assessed at the level of individual cells, while other characteristics are typically assessed based on the whole sample. It will be appreciated that a cell line may be considered to possess a characteristic even if the characteristic is not present or detected in all cells of a sample. For example, depending on factors such as the particular characteristic and method of assessment, at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the cells may exhibit the characteristic in various embodiments of the invention.

[00101] In some embodiments of the invention, a characteristic of an ES cell line remains stable for at least 5 weeks in culture, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 weeks, or more (during which time the cells are passaged as appropriate). In some embodiments of the invention, a characteristic of a cell line remains stable for at least 10 passages, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100 passages, or more. A characteristic is considered "stable" if the cell line continues to exhibit the characteristic without substantial change, e.g., the characteristic is essentially unchanged over a time period of interest. For example, a cell line that is "positive" for expressing a particular marker remains positive. It will be understood that the proportion of cells in a sample that exhibit a particular

characteristic may differ somewhat among different samples and/or when assessed multiple times. In some embodiments, if a characteristic is stable, such characteristic does not exhibit a trend towards a substantial change when assessed at different time points. In some embodiments, a substantial change is a change (increase or decrease) of more than 20%) relative to an initial value. In some embodiments a substantial change is a change of more than 50%) relative to an initial value. In some embodiments a substantial change is a change of more than 1.5-fold relative to an initial value.

[00102] In some embodiments, an ESC line has a normal karyotype. In general, an ES cell has a normal karyotype if the cell has the normal complement of chromosomes for a diploid cell of that species and there are no evident chromosomal abnormalities such as

translocations, inversions, or deletions. Karyotype can be assessed using, e.g., G-banding or FISH. In certain embodiments, greater than 75%, 80%, 85%, 90%, 95%, or more than 95% of cells examined display a normal karyotype. In some embodiments at least 20 cells are examined. In some embodiments of the invention an ESC line is male (has one X and one Y chromosome). In some embodiments, an ESC line is female (has two X chromosomes).

[00103] An ESC line of the invention is positive for at least one master pluripotency transcription factor such as OCT4 (also known as POU5F1), SOX2, and/or NANOG. In some embodiments, an ESC line is positive for all three of these factors. An ESC line of the invention is positive for at least one extracellular surface pluripotency marker. For example, a primate ES cell line, e.g., an hESC line, is typically positive for SSEA4 and TRA1-60. A primate ES cell line of the invention can be positive for other markers such as, e.g., SSEA3, TRA-1-81 , GCTM2, GCT343, CD9, Thy 1 , tissue-nonspecific alkaline phosphatase, and/or class 1 HLA.

[00104] In some embodiments, an ESC line is negative for markers of the three germ layers (endoderm, mesoderm, ectoderm) and is negative for multiple lineage-specific markers. A lineage-specific marker is a marker characteristically expressed only or substantially only by cells of particular lineage(s) and is useful for identifying cells of those lineage(s). For example, an ESC line can be negative for expression products of neural lineage genes (e.g., nestin), hematopoietic lineage genes (e.g., GATA-1), muscle lineage genes (e.g., MEF genes), etc. In some embodiments, an ESC line is negative for PAX6, OLIG2, NeuroDl, SerpinAl , AFP, PTF1A, and/or FOXA2.

[00105] In some embodiments, a characteristic of interest relates to X chromosome inactivation status, e.g., in a female ESC cell or cell line. Such characteristics include level of XIST expression, exclusion of COT-1 RNA from the non-pseudoautosomal region of the X chromosome, methylation status of the XIST promoter region, histone modification status of the X chromosome (e.g., presence or absence of repressive histone modifications such as H3 27me3 along an X chromosome), presence or absence of XIST foci or clouds coating at least part of one X chromosome, monoallelic or biallelic expression of X-linked gene(s). Portions of the X chromosome (e.g., pseudoautosomal region(s)) normally escape X inactivation. Therefore, most assays relating to determining the X inactivation status involve analyzing portions of the X chromosome other than the pseudoautosomal region, although the pseudoautosomal region may be analyzed as a control representing a non-inactivated state.

[00106] As described in detail in the Examples, use of physiological 02 conditions allowed the derivation of hESC lines with two active X chromosomes (XaXa hESCs), which underwent XCI upon differentiation or following shift to atmospheric 02 conditions. Notably, XCI was random, as occurs during normal mammalian development, resulting in a population of hESCs that exhibits biallelic expression of X-linked genes. In contrast, hESCs derived under atmospheric 02 conditions exhibit non-random XCI, resulting in a population of cells that exhibits monoallelic expression of X-linked genes. In certain aspects, the invention provides female primate ESC lines, e.g., human ESC lines, that are pre-X- inactivation. In some embodiments of particular interest, a female ESC line of the invention exhibits low or undetectable XIST expression. In some embodiments, XIST expression (e.g., as measured using RT-PCR, optionally normalized to a housekeeping gene such as GAPDH) is undetectable (e.g., does not differ in a statistically significant manner from background levels). In some embodiments, the level of XIST expression is no more than 1%, 5%, 10%, or 25% of that of a control level. In some embodiments, the control level is the level is that exhibited by the same ESC line when maintained under atmospheric 02 conditions for at least 2 weeks. In some embodiments, the control level is the level exhibited by the WIBR2 or WIBR3 cell line that was derived under physiological 02 conditions and has been maintained under atmospheric 02 conditions for at least 2 weeks. In some embodiments, a female hESC line exhibits no or essentially no XIST positive cells (e.g., the percentage of XIST positive cells detected does not differ in a statistically significant manner from background levels or is no more than 5%). RNA FISH using a probe that hybridizes to XIST can be used to perform such assays. In some embodiments, RNA FISH is performed using COT-l DNA as a probe. Inactivation of the X chromosome is indicated by exclusion of COT- 1 RNA from the non-pseudoautosomal region of the chromosome. If desired, DNA FISH can be performed as well to confirm that the cell has contains two X chromosomes. In some embodiments, a female ESC line exhibits at least 90% CpG methylation at the XIST promoter. In some embodiments, a female ESC line of the invention exhibits biallelic expression of at least 30% of X-linked genes, e.g., at least 40% or at least 50% of X-linked genes, indicating that the line has two active X chromosomes (XaXa). In some embodiments, a female ESC line of the invention exhibits biallelic expression of at least 60%, 70%, 80%, 90% or more of X-linked genes. Whether a gene is biallelically expressed can be assessed, e.g., by examining expression at one or more informative variations, e.g., single nucleotide polymorphisms (SNPs) within the gene (which can be performed on a population of cells) or by performing RNA FISH to examine transcription of an X-linked gene. An informative variation, e.g., an informative SNP, is a variation wherein the two chromosomes differ with regard to DNA sequence at a particular location (e.g., a single nucleotide in the case of a SNP), i.e., the two X chromosomes contain different alleles. Biallelic expression of an informative X-linked SNP can thus serve as an indicator that the gene containing the SNP is biallelically expressed. Methods for assessing allele-specific expression are known in the art and include, e.g., hybridization to allele specific oligonucleotide probes (e.g., on an array), allele-specific primer extension, reverse-transcription PCR using allele-specific primers followed by detection (e.g., by mass spectrometry), reverse transcription followed by sequencing, etc. In some embodiments, at least 10-20 different genes outside the

pseudoautosomal region are assessed. In some embodiments, an ESC line is considered to exhibit biallelic expression of an X-linked gene if the ratio of the expression of a first allele to the expression of the other allele is not greater than 3: 1. For example, the ratio of expression levels may be between 2: 1 and 1 :2, or about 1 : 1. Stated another way, if between 25% and 75% of the transcripts, e.g., between 33% and 66%, e.g., about 50% of the transcripts originate from a first allele while the remaining transcripts originate from the other allele, the gene is considered to be expressed biallelically. RNA FISH allows examination of expression within individual cells. If two foci are seen within a cell, the gene is biallelically expressed by that cell, while observing only a single focus indicates that the gene is probably expressed monallelically by that cell. In some embodiments, a cell line is considered to be XaXa if biallelic expression of 10-20 or more X-linked genes outside the pseudoautosomal region is seen in at least 50% of the cells in a sample, as assessed by RNA FISH.

[00107] In some embodiments, a female ESC line of the invention is negative for XIST expression, is XaXa, and retains the ability to induce XIST upon (i) differentiation; (ii) culture under atmospheric 02 conditions; and/or (iii) exposure to cell stresses such as Hsp90 inhibition, proteasome inhibition, oxidative stress due to oxidizing agents or inhibitors of endogenous anti-oxidant systems, or harsh freeze-thaw cycles. Induction of XIST can be assessed, e.g., by qRT-PCR or by RNA FISH. In some embodiments, XIST transcript levels increase by at least 10, 50, 100 fold or more. In some embodiments, the ratio of XIST RNA to GAPDH mRNA increases from less than 0.005 to between 0.02 and 3. In some embodiments, XIST expression results in foci or clouds of XIST along the X chromosome from which XIST is expressed which can be observed, e.g., using RNA FISH to visualize XIST and, if desired, DNA FISH to visualize the X chromosome. [00108] In some embodiments, a female ESC line of the invention is XaXa and is capable of undergoing XCI, e.g., upon (i) differentiation; (ii) culture under atmospheric 02 conditions; and/or (iii) exposure to cell stresses such as Hsp90 inhibition, proteasome inhibition, oxidative stress due to oxidizing agents or inhibitors of endogenous anti-oxidant systems, or harsh freeze-thaw cycles. In some embodiments, expression of XIST serves as an indicator that XCI has occurred. For example, presence of XIST clouds along one of the two X chromosomes indicates that XCI has occurred. Accumulation of a repressive histone modification such as H3K27 trimethylation on one of the X chromosomes is another indicator of XCI. In some embodiments, whether XCI has occurred is determined by assessing expression of X-linked genes outside the pseudoautosomal region in individual cells. For example, expression can be assessed using RNA FISH, wherein presence of two foci indicates that XCI has not occurred, whereas presence of only a single focus indicates that XCI has likely occurred. Single cell RT-PCR of informative X-linked transcripts could also be used to determine whether XCI has occurred. In embodiments of particular interest a female ESC line of the invention is is XaXa and is capable of undergoing random X inactivation, e.g., upon (i) differentiation; (ii) culture under atmospheric 02 conditions;

and/or (iii) exposure to cell stresses such as Hsp90 inhibition, proteasome inhibition, oxidative stress due to oxidizing agents or inhibitors of endogenous anti-oxidant systems, or harsh freeze-thaw cycles. In some embodiments, biallelic expression of at least 30% of X- linked genes outside the pseudoautosomal region after XCI indicates that random XCI has occurred. For example, in some embodiments the ESC line exhibits biallelic expression of at least 50% of X-linked genes outside the pseudoautosomal region after XCI. In some embodiments, an XaXi ESC line is considered to exhibit biallelic expression of an X-linked gene if the ratio of the expression of a first allele to the expression of the other allele is not greater than 3: 1. For example, the ratio of expression levels may be between 2: 1 and 1 :2, or about 1 : 1. Stated another way, if between 25% and 75% of the transcripts, e.g., between 33% and 66%, e.g., about 50% of the transcripts originate from a first allele while the remaining transcripts originate from the other allele, the gene is considered to be expressed biallelically. In some embodiments, an XaXi ESC line is considered to exhibit biallelic expression of an X-linked gene if between 30% and 70%, e.g., between 40% and 60%, e.g., about 50% of the cells express a first allele of the gene (and do not express the second allele), and the majority of the other cells express the other allele (and do not express the first allele). [00109] As described herein, derivation under physiological 02 conditions allowed the isolation of hESCs with two active X chromosomes (XaXa hESCs), which underwent XCI upon differentiation or following shift to atmospheric 02 conditions. Notably, XCI was random, as occurs during normal mammalian development, resulting in a population of cells that exhibits biallelic expression of X-linked genes. In contrast, hESCs derived under atmospheric 02 conditions exhibit non-random XCI, resulting in a population of cells that exhibits monoallelic expression of X-linked genes. Thus, hESCs of the invention can be differentiated into cell populations that exhibit biallelic expression of X-linked genes.

Without wishing to be bound by any theory, biallelic expression of X-linked genes may have advantages for potential therapeutic or other applications such as models for disease. For example, a cell population exhibiting biallelic expression may more closely replicate the situation existing in vivo in female mammals. In addition, hESCs derived under

physiological 02.conditions exhibited a number of other notable characteristics. For example, and without limiting the invention in any way, it was observed that hESCs derived under physiological 02 were generally easier to culture, had a higher cloning efficiency (e.g., up to about passage 10), exhibited less propensity to spontaneously differentiate while being maintained in hESC maintenance medium, and were more amenable to differentiation under differentiating conditions than were hESCs derived under atmospheric 02 conditions.

Without wishing to be bound by any theory, derivation under physiological 02 may result in primate ESCs that are less mature than prior art primate ESCs. For example, derivation under physiological 02 may result in hESCs that are less mature than prior art hESCs.

[00110] The invention encompasses the recognition that at least some characteristics of ESCs derived under physiological 02 conditions may be present in ESCs that have had limited exposure to non-physiologic 02 conditions during derivation. In some aspects, the invention provides ES cell lines, cells, and cell populations derived substantially under physiological 02 conditions. Derivation "substantially under physiological 02 conditions" encompasses derivation under physiological 02 conditions as described above, but in certain embodiments permits limited exposure to non-physiological 02 conditions, e.g., atmospheric 02 conditions. In some embodiments, "substantially under physiological 02 conditions" encompasses short exposures to non-physiological 02 conditions, e.g., lasting from less than 1 minute up to about 5-10 minutes, during the derivation process. In some embodiments, "substantially under physiological 02 conditions" encompasses somewhat longer exposures to non-physiological 02 conditions, e.g., lasting from about 10-60 minutes. In some embodiments, any such exposure(s) occur no more than once per 24 hours during the derivation period. In certain embodiments, after being placed in derivation media, the embryo or portion thereof and proliferating cells are maintained under physiological 02 conditions for at least 95%, 98%, 99%, 99.5%, 99.9% or more of the derivation period, and any exposures to non-physiological 02 conditions last no more than 60 minutes and, in some embodiments, occur not more than once per 24 hours. In certain embodiments, the embryo or portion thereof and proliferating cells are maintained within a desired range of physiological 02 concentrations for at least 95%, 98%, 99%, 99.5%, 99.9% or more of the derivation period, and any exposures to non-physiological 02 conditions last no more than 60 minutes and, in some embodiments, occur not more than once per 24 hours. In some embodiments, such exposure(s) (i) do not cause a substantial change in the XCI status of an XaXa ES cell line; (ii) do not substantially decrease the efficiency of deriving XaXa ES cell lines; and/or (iii) do not cause a substantial change in the gene expression profile with respect to a set of at least 10 genes whose expression is upregulated or downregulated in ESCs following acute exposure to atmospheric 02 conditions.

[00111] In some embodiments, a substantial change in gene expression profile refers to at least a 2-fold change in expression level of at least 20% of the genes assessed. In some embodiments, whether an exposure to non-physiological 02 conditions causes a significant change in gene expression profile is determined by comparing the gene expression profile of an ES cell line derived under conditions that include the exposure with that of a genetically matched ES cell line that was derived under conditions that are otherwise essentially identical but do not include such exposure. In some embodiments, whether an exposure to non- physiological 02 conditions causes a significant change in XCI status is determined by determining whether the exposure is sufficient to cause XCI to occur in an XaXa ES cell line. In some embodiments, whether an exposure to non-physiological 02 conditions causes a significant change in gene expression profile is determined by subjecting a population of ES cells derived and maintained under physiological 02 conditions to such exposure and determining whether a significant change in gene expression profile occurs as compared with a genetically matched population of ES cells. For example, a first gene expression profile can be obtained from a first sample of cells from said population that has not undergone such exposure. A second gene expression profile is obtained from a second sample of said cells that has undergone such exposure, and the two gene expression profiles are compared.

Exemplary genes whose expression is upregulated or downregulated in hESCs following acute exposure to atmospheric 02 conditions are listed in Table S2.

[00112] In some embodiments, a substantial decrease in efficiency of deriving XaXa ES cell lines refers to a decrease of at least 50%. In some embodiments, reduction of CpG methylation of the XIST promoter to 50%-60% consistent with one XIST allele having been activated, increase in XIST expression, and/or appearance of XIST foci or clouds indicates a substantial change in XCI status.

[00113] ESC Post-derivation Maintenance

[00114] In some embodiments of the invention, ESCs and ESC lines of the invention derived under or substantially under physiological 02 (e.g., ESC lines at passage 2 or greater) continue to be maintained under such conditions following derivation. In some

embodiments, such ESC lines are maintained for at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more passages under or substantially physiological 02 conditions. In some embodiments, an ESC line exhibits a stable karyotype over at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more passages. In some embodiments, an ESC line exhibits a stable XaXa phenotype over at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more passages. For example, the line remains negative for XIST expression, exhibits over 90% CpG methylation at the XIST promoter, and/or exhibits biallelic expression of X-linked genes. In some embodiments the line retains the ability to activate XIST expression and undergo XCI, e.g., random XCI, over at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more passages.

[00115] ESCs of the invention are maintained in a suitable culture medium, which may differ from that used for ESC derivation. In some embodiments, primate ESCs, e.g., hESCs, are maintained in medium that is free or essentially free of LIF. ESCs can be passaged mechanically or enzymatically (e.g., using collagenase, dispase, or trypsin). In some embodiments, mechanical passaging is used over at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more passages. ESCs can be frozen for long-term storage, e.g., in liquid N2. In some embodiments, mechanical passaging is used for initial passages, e.g., between 1 -10 passages, and enzymatic passaging is used for at least some subsequent passages. In certain

embodiments of the invention an ESC line retains a normal karyotype when frozen and thawed. In certain embodiments of the invention an ESC line retains an XaXa phenotype and the ability to undergo XCI, e.g., random XCI, when frozen and thawed. [00116] Genetic Modification

[00117] In some aspects, the invention provides genetically modified ESCs derived under physiological 02 conditions. In some embodiments, a "genetically modified ESC" is an ESC that has an alteration in its genome that has been produced by the hand of man in the cell or in an ancestor of the cell. In some embodiments, an ESC is genetically modified by introducing exogenous genetic material into the cell, wherein at least some of the exogenous DNA may become inserted into the genome of the cell. As used herein, exogenous DNA introduced by the hand of man is referred to as a "transgene". In some embodiments, inserted DNA replaces a segment of endogenous genomic DNA. The term "transgene" also encompasses modified endogenous DNA, such as DNA that has a deletion engineered by the hand of man, wherein the resulting DNA sequence differs from that present in the cell prior to the intervention leading to the deletion. Genetic modifications can include, e.g., physical removal of all or a part of a gene and/or insertion of a nucleic acid into a gene (e.g., into an exon), which may functionally inactivate the gene (also referred to as knocking out the gene).

[00118] In some aspects, an ESC is genetically modified using a suitable vector. For example, an appropriate vector (e.g., a plasmid or virus) can be used to introduce a nucleic acid into cells in order, for example, to integrate DNA into genomic DNA, express introduced DNA in recipient cells, cause recombination (homologous or nonhomologous) between introduced DNA and endogenous DNA and/or knock out endogenous gene(s).

Suitable virus vectors include, e.g., adenoviruses, adeno-associated viruses, retroviruses (e.g., lentiviruses), etc. In some embodiments other approaches, such as exposing a cell to a chemical such as a mutagenic compound that can alter DNA, can be used. As used herein, altered DNA, such as exogenous DNA introduced by the hand of man is referred to as a transgene. The exogenous genetic material, also referred to as a "transgene" can comprise or consist of a nucleic acid having any sequence of interest. In some embodiments, the sequence encodes a polypeptide or RNA of interest. In some embodiments, the inserted DNA further comprises one or more regulatory elements e.g., expression control elements such as a promoter or promoter/enhancer which may be operably linked to the coding sequence. Other genetic elements such as a terminator, polyadenylation site, IRES sequence, etc., can be present. In some embodiments, inserted DNA (e.g., a coding sequence) is inserted into the genome such that its expression is controlled by endogenous regulatory elements, e.g., expression control elements such as a promoter, enhancer, etc. In some embodiments the transgene comprises tissue-specific or cell-type specific expression control elements, so that the gene is expressed selectively in one or more cell types or tissues relative to others. In some embodiments the gene comprises regulatable expression control element(s), e.g., an inducible or repressible promoter. Examples of regulatable promoters include heat shock promoters, metallothionein promoter, and promoters that comprise an element responsive to a small molecule such as tetracycline or a related compound (e.g., doxycycline), or a hormone. In some embodiments the cell expresses appropriate transacting protein(s), e.g., a protein comprising a DNA binding domain, activation or repression domain, and ligand-binding domain to render transcription responsive to a ligand.

[00119] A transgene can encode any of a wide variety of polypeptides or RNAs. In some embodiments the transgene encodes a marker protein, reporter, or genetically encoded sensor, e.g., one that would allow detection of one or more cell types or detection of a process or event or metabolite, etc. Exemplary marker proteins include fluorescent proteins such as green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and fluorescent variants such as enhanced GFP (eGFP), mCherry, etc., and luminescent or luminescence-producing proteins such as luciferase (e.g., firefly or Renilla luciferase), aequorin. In some embodiments the transgene encodes a fusion protein that comprises a polypeptide of interest and a marker protein. In some embodiments the transgene encodes a fusion protein that comprises a polypeptide of interest and a tag, e.g., an epitope tag that can be conveniently used for detection or purification. Λ transcriptional reporter could comprise a nucleic acid encoding a marker protein wherein the nucleic acid is operably linked to promoter of interest. In some embodiments a transgene encodes a short hairpin RNA, microRNA precursor, or antisense RNA. In some embodiments, conditional targeting is used, which allows tissue-specific and/or inducible inactivation of a gene. In some embodiments, a transgene comprises recognition sites for a recombinase. Introducing the recombinase or inducing its expression in the cell can be used to remove genetic material between the recognition sites. Such removal could induce expression of the gene, e.g., by bringing a coding sequence into operable association with expression control elements, removing a stop codon or other blocking sequence, etc. Thus tissue-specific and/or inducible inactivation or expression of a gene into which recombination recognition sites have been introduced can be achieved by controlling the expression of the recombinase. In other embodiments, the recombinase could remove at least part of the introduced DNA, e.g., after selection. Useful recombinase systems include the Cre/lox, bacteriophage lambda, and Frt/flp systems. A genetic modification in non-human ES cells can be chosen to produce a phenotype that is similar to (mimics) a condition that occurs in other species (e.g., humans), e.g., to produce a model for that condition. In other embodiments a modification can be chosen to modify a phenotypic characteristic of an animal. In some embodiments a genetic modification "repairs" a mutation or defect, e.g., by homologous recombination. In some embodiments a genetic modification comprises a gene that encodes a therapeutically beneficial protein or RNA or functionally inactivates a gene that encodes a deleterious protein (e.g., a mutated or activated oncogene). In some embodiments, ESCs of the invention have multiple genetic modifications.

[00120] In some embodiments, homologous recombination is used to target a transgene to a desired location in the genome, e.g. resulting in site-specific gene addition, knockout, or other modification. Any means known in the art to generate cells with targeted integration can be used. To facilitate the selection of homologous recombination events over the nonhomologous recombination events, at least two enrichment methods have been developed: the positive-negative selection (PNS) method and the "promoterless" selection method. Briefly, PNS, the first method, is in genetic terms a negative selection: it selects against

recombination at the incorrect (non-homologous) loci by relying on the use of a negatively selectable gene that is placed on the flanks of a targeting vector. On the other hand, the second method, the "promoterless" selection, is a positive selection in genetic terms: it selects for recombination at the correct (homologous) locus by relying on the use of a positively selectable gene whose expression is made conditional on recombination at the homologous target site. See, e.g., Mortensen R., Curr Protoc Mol Biol. Chapter 23 :Unit 23.1 , 2006 for description of mammalian gene targeting in the context of mouse cells. See also Waldman T, et al., Human somatic cell gene targeting. Curr Protoc Mol Biol. Chapter 9:Unit 9.15, 2003. Rago C, Genetic knockouts and knockins in human somatic cells. Nat Protoc. 2(1 1):2734- 46, 2007. In some embodiments, a transgene is targeted to a "safe harbor locus" in the genome. In some embodiments, a safe harbor locus is a site whose disruption does not have any known significant or deleterious effect on mammalian cells. In some embodiments the site is present in a single copy in the genome of a mammal of interest. In some embodiments the locus encodes a protein or a non-coding RNA with no known function in mammalian cells. In some embodiments, the site is constitutively expressed, demonstrating

transcriptional competence to maintain stable, uniform expression of a gene of interest. An exemplary safe harbor locus is the preferred site of integration of the adeno-associated virus (AAVS1) on human chromosome 19. In some embodiments, a safe harbor locus has been validated in the mouse by generating a transgenic mouse in which the locus is knocked out and showing that the knockout mouse is viable and healthy, showing no evident signs of disease. An exemplary validated locus is the Rosa26 locus. .See, e.g., Irion, S. et al.

Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat.

Biotechnol. 25, 1477-1482 (2007). See, e.g., Costa, M. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792-796 (2007); Suzuki, K. et al. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc. Natl. Acad. Sci. USA 105, 13781-13786 (2008); and Zwaka, T.P. & Thomson, J. A. Homologous recombination in human embryonic stem cells. Nat. Biotechnol. 21 , 319-321 (2003) for examples of gene targeting in hESCs.

[00121] In some aspects, an ESC is genetically modified using a zinc finger nuclease (ZFN). This technique involves introducing DNA double-strand breaks by site-specific ZFNs to facilitate homologous recombination. Fokl is an exemplary C2H2 ZFN that can be used. A ZFN can be generated by fusing the nuclease domain of a ZFN, e.g., the Fokl nuclease domain, to a DNA recognition domain composed of engineered C2H2 zinc-finger motifs that specify the genomic DNA binding site for the chimeric protein. Upon binding of two such fusion proteins at adjacent genomic sites, the nuclease domains dimerize, become active and cut the genomic DNA. When a donor DNA that is homologous to the target on both sides of the double-strand break is provided, the genomic site can be repaired by homology-directed repair, allowing the incorporation of exogenous sequences placed between the homologous regions. See, e.g., PCT/US2003/009081 (WO/2003/080809); Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646-651 (2005); Carroll, D. Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther. 15, 1463-1468 (2008); Moehle, E.A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 104, 3055-3060 (2007); Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298-1306 (2007). In some embodiments zinc finger DNA-binding domains with alterations in at least one zinc coordinating residue, such as CCHC zinc fingers. See, e.g., PCT/US2007/025455 (WO/2008/076290).

[00122] II. Compositions and Methods for Protecting Stem Cells from Cell Stress

[00123] The invention encompasses the recognition that primate, e.g., human, ESC X chromosome activation state is highly sensitive to environmental conditions. For example, it was found that oxidative stress, or cell stress caused by proteasome inhibition, or harsh freeze-thaw cycles could cause XCI to occur. Such conditions may prevent XaXa ESCs from properly undergoing XCI upon differentiation. The invention provides compositions and methods useful for protecting ESCs from cell stress.

[00124] "Cell stress" generally refers to a situation in which cells are exposed to a condition that causes damage to one or more cellular biomolecule(s) or structure(s) or otherwise inhibits the ability of such molecule(s) or structure(s) to function properly. The condition(s) causing cell stress can be physical (e.g., freezing/thawing) or chemical (e.g., exposure to a compound). "Oxidative stress" refers to cell stress associated with oxidation of cellular biomolecule(s) such as nucleic acids (e.g., DNA), proteins, or lipids. Oxidative stress can be caused by free radicals or reactive oxygen species (ROS) and can arise, e.g., from exposure to such compounds or from an imbalance between their generation and elimination. "Free radical" has been defined as "any species capable of independent existence that contains one or more unpaired electrons" (Halliwell, B. & Gutteridge, J. Free Radicals in Biology and Medicine, 4^th ed., Oxford University Press, 2007). In some aspects, a free radical is any atom or molecule that has a single unpaired electron in an outer shell. Some ROS (but not all) are free radicals. ROS are reactive molecules that contain one or more oxygen atoms. Examples of ROS include, O₂- (superoxide anion), hydrogen peroxide (H202), hydroxyl radical (ΟΗ·), alkoxy radicals (RO●), peroxy radicals (ROO^»),

hypochlorous acid (HOC1), and peroxynitrite (ONOO-). Other reactive compounds that can cause cell stress include reactive nitrogen species, reactive chlorine species, and reactive bromine species (Halliwell, B. & Gutteridge, supra).

[00125] In some aspects, the invention provides a method comprising deriving and/or maintaining an ES cell line under conditions that reduce cell stress. In some embodiments, an ESC line is derived under conditions that reduce cell stress and is subsequently maintained under conditions that reduce cell stress. The invention encompasses a variety of methods of reducing cell stress, e.g., oxidative stress. One method comprises culturing ESCs under physiological 02 conditions. As described herein, maintaining XaXa ESCs under physiological 02 conditions preserved their XaXa status. Other methods comprise culturing ESCs under conditions that mimic the effects of physiological 02 conditions (or,

equivalently, inhibit or antagonize the effect of supra-physiological 02 conditions). In some aspects, physiological 02 conditions can be mimicked using chemical compounds. It was also observed that the XaXa status of XaXa ESCs derived under physiological 02 conditions could be preserved under atmospheric 02 conditions by maintaining the cells in medium comprising any of a variety of antioxidants.

[00126] The invention provides a method of deriving a cell line comprising steps of: (a) obtaining an isolated early stage embryo; and (b) deriving a pluripotent ES cell line from the isolated early stage embryo under conditions that mimic the effect of physiological 02 conditions. In some embodiments, the embryo is a primate embryo, e.g., a human embryo. In some embodiments, the conditions that mimic the effect of physiological 02 conditions comprise using culture medium comprising at least one compound that mimics the effect of physiological 02 conditions. In some embodiments, the culture medium comprises at least one antioxidant. An "antioxidant" can be defined as "any substance that delays, prevents or removes oxidative damage to a target molecule" (Halliwell, B. & Gutteridge, supra). A target molecule can be any oxidizable substrate, e.g., any organic molecule found in vivo. In some embodiments, an antioxidant is a substance that significantly delays or prevents oxidation of an oxidizable substrate. In some embodiments, an antioxidant is capable of significantly delaying or preventing oxidation of an oxidizable substrate even when present at low concentrations compared with those of an oxidizable substrate. For purposes of description, a compound that reduces the activity of ROS, RNS, and/or other similarly reactive species is considered an antioxidant. In some embodiments, an antioxidant is a free radical scavenger. "Free radical scavenger" can be any compound that reduces the reactivity of a free radical. In some aspects, a free radical scavenger reacts with a free radical, such that the resulting product(s) have reduced reactivity as compared with that of the free radical. In some aspects, resulting product(s) contain fewer unpaired electrons than present in the free radical, e.g., they do not contain an unpaired electron. Antioxidants encompass compounds that promote activity of endogenous antioxidant systems, e.g., compounds that promote activity of endogenous enzymes with antioxidant activity, or that promote activity of other endogenous compounds having antioxidant activity. For example, such compounds may be used in mammalian cells to regenerate a species with antioxidant activity, may function as an enzyme cofactor, or may induce synthesis of an endogenous enzyme or other compound with antioxidant activity or function as a cofactor for such an enzyme. Antioxidants also encompass compounds that inhibit synthesis or activity of an endogenous enzyme that generates free radicals or other reactive species in vivo or sequester cofactors for such enzyme(s). An antioxidant could have multiple activities. For example, it may both scavenge free radicals and inhibit a free radical generating enzyme such as xanthine oxidase. In some embodiments, an antioxidant is a compound that inhibits one or more of the following: (i) lipid peroxidation; (ii) DNA oxidation; (iii) oxidation of amino acids in proteins; (iv) oxidation of a cofactor for an enzyme expressed in the early mammalian embryo and/or in ESCs.

[00127] In some embodiments, an inventive method comprises inhibiting formation of free radicals and/or ROS during the derivation or subsequent maintenance or differentiation of ESCs. In some embodiments the invention provides a method comprising deriving or maintaining ESCs in medium containing an antioxidant that reacts with one or more free radicals or ROS and substantially reduces its reactivity towards cellular biomolecules. In some aspects, the invention comprises inducing expression or enhancing the activity of a cellular enzyme such as a superoxide dismutase, catalase, glutathione peroxidase or peroxiredoxin that help protect cells against oxidative stress. Such enzymes may convert ROS to less reactive gaseous oxygen and water molecules. In some embodiments, the method comprises providing a compound that induces expression or acts as a cofactor for such enzyme(s). In some embodiments, the invention comprises inducing a protective response in the cell that can help counteract cell stress and/or repair stress-induced damage or dysfunction.

[00128] Antioxidants can be, e.g., small molecules, lipids, or proteins. As used herein, the term "antioxidant" includes compounds with a known structure as well as compositions that may be at least partially uncharacterized. An antioxidant can be a naturally occurring compound or may be a compound invented by man. Exemplary antioxidants include vitamin C, vitamin E, lipoic acid, L-sulforaphane, reduced L-glutathione, butylated hydroxyanisole, alpha tocopherol, deferoxamine, resveratrol, N-acetylcysteine, Trolox, and morin hydrate. As described in Example 8, addition of these compounds to hESC culture medium preserved the XaXa status of female hESCs derived under physiological 02 conditions.

[00129] In some embodiments, the antioxidant comprises vitamin C. "Vitamin C" encompasses ascorbic acid and salts of ascorbic acid (e.g., calcium ascorbate, sodium ascorbate, and other ascorbate salts). As used herein, any of these compounds, or

combinations thereof, is considered "vitamin C". A vitamin C can contain a selected proportion or ratio of ascorbic acid and/or salts thereof.

[00130] in some embodiments, the antioxidant comprises vitamin E. "Vitamin E" encompasses alpha, beta, gamma, and delta tocopherol and alpha, beta, gamma, and delta tocotrienol. As used herein, any of these compounds, or combinations thereof, are considered "vitamin E". Vitamin E can contain a selected proportion or ratio of tocopherol(s) and/or tocotrienol(s). In some embodiments of the invention, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the tocopherol/tocotrienol content of a vitamin E by weight consists of a particular tocopherol or tocotrienol. For example, in embodiments, at least 50% of the tocopherol/tocotrienol content by weight is alpha tocopherol.

[00131] In some embodiments, the antioxidant comprises lipoic acid. Lipoic acid exists as two enantiomers R-(+)-lipoic acid (RLA) and S-(-)-lipoic acid (SLA) and as a racemic mixture R/S-lipoic acid (R/S-LA). Either enantiomer, or mixtures thereof, can be used in various embodiments of the invention.

[00132] In some embodiments, the antioxidant comprises morin, also referred to as morin hydrate. Morin hydrate is a flavonoid that can be isolated from a variety of plants such as Madura pomifera (Osage orange), Maclura tinctoria (old fustic) and from leaves of Psidium guajava (common guava).

[00133] In some embodiments, the antioxidant comprises curcumin. "Curcumin"

(diferuloylmethane) is a polyphenolic compound that is a component of the Indian spice turmeric and can be isolated from the turmeric plant (Curcuma longa). Commercially available preparations of curcumin typically also contain the structurally related compounds demethoxycurcumin (e.g., -17%) and bisdemethoxycurcumin (e.g., ~3%). See, e.g., Aggarwal, BB, Trends Pharmacol Sci. 30(2):85-94, 2009. The invention encompasses embodiments in which purified curcumin or a mixture comprising curcumin and additional curcuminoids is used. [00134] In some embodiments, an antioxidant is selected from among the following nonlimiting group of compounds: vitamin A, a vitamin B (e.g., vitamin B l , B2, B6, and/or B12), coenzyme Q, green tea (epigallocatechin gallate-EGCG), citric acid, oxalic acid, phytic acid, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, gallic acid, gallotannins, rosmarinic acid, silymarin, eugenol, manganese, zinc, adenosine, transferrin, lactoferrin, cysteine, histidine-containing dipeptides, pyridoxamine, carotenoids, flavonoids (e.g., kaempferol, myricetin, silymarin, quercetin), flavones and flavonols (e.g., planar flavones and flavonols with a 7-hydroxyl group), other phenolic compounds (e.g., plant-derived phenolics), melatonin, coelenterazine, nordihydroguaiaretic acid (NDGA), curcumin

(diferuloylmethane), 21 -amino steroids (also called lazaroids), various antibiotics (e.g., tetracyclines), azoles (e.g., ketoconazole), thiols (e.g., mercaptopropionylglycine), metal ion chelators (e.g., iron chelators such as ICRF-187, deferasirox, deferiprone, hydroxypyridones), fullerenes, xanthine oxidase inhibitors (e.g., purine analogues such as allopurinol, oxypurinol, and tisopurine and other compounds such as febuxostat and inositols), selenium (e.g., sodium selenite, selenous acid), uric acid, bilirubin, trehalose, lipid-soluble chain-breaking

antioxidants such as BHT and ethoxyquin, superoxide dismutase (SOD), catalase, superoxide dismutase (SOD)/catalase inducers or mimetics such as EUK-134, EUK-189, or EUK-207 (see, e.g., Lui, R., et al., Proc Natl Acad Sci U S A.100(14): 8526-8531 , 2003), NXY-059 (U.S. Pat. Pub. No. 20030181527), penicillamine, bucillamine, salicyclic acid,

aminosalicylates, apomorphine, selegeline, flupiritine, omeprazole, 4-hydroxytamoxifen, certain ACE inhibitors, probucol, propofol, certain beta blockers (e.g., metoprolol),certain calcium channel blockers, phenylbutazone, troglitazone, tacrolimuns, idebenone, nitrecapone, entecapone, and edaravone. Oxidative stress may play a role in a variety of diseases, including neurodegenerative diseases, atherosclerosis, diabetes, rheumatoid arthritis and other inflammatory diseases, stroke, myocardial infarction, and ischemia-reperfusion injury. In some embodiments of the invention, an antioxidant used in the derivation and/or maintenance of ESCs is a compound that has been developed or tested for potential use in one or more of these disease(s).

[00135] In some embodiments, an antioxidant is a derivative of a compound referred to above. In this regard, a "derivative" is a compound that is or can be derived from a structurally similar compound or a compound that can be imagined to arise from a first compound if one or more atom(s) of the first compound is/are replaced with another atom or group of atoms, provided that the resulting compound retains significant structural and/or functional similarity to the first compound. For example, derivatives may share a common core structure or scaffold (e.g., a common pharmacophore). One of skill in the art of medicinal chemistry will be aware of many common methods for generating derivatives. For example, compounds with a free hydroxyl group may readily be converted to esters.

Bioisosteric replacement can be used, wherein a substituent or group is replaced with a different substituent or group with similar physical or chemical properties and that imparts similar (but sometimes improved) biological properties to a chemical compound.

Derivatives may also be referred to as "analogs". Derivatives of a number of the above- mentioned compounds are known in the art. See, e.g., U.S. Pat. Pub. No. 20100063125 (melatonin derivatives), Dubuisson ML, et al., Drug Dev Ind Pharm.; 31(9):827-49, 2005 (coelenterazine derivatives); Gurkain, AS, et al, Archiv der Pharmazie, 338 (2-3): 67 - 73, 2005 (lipoic acid derivatives), Aggarwal, supra; Fuchs JR, Bioorg Med Chem Lett.,

19(7):2065-9, 2009 (curcumin derivatives) for some examples. In some embodiments, the activity of a derivative (e.g., antioxidant activity or free radical scavenging activity) is at least 10% of that of the compound of which it is a derivative, e.g., between 10% and 100%. In some embodiments, the antioxidant activity of a derivative is greater than that of the compound of which it is a derivative, e.g., by a factor of between 1 and 100. In some embodiments a derivative has a desired feature, such as increased water solubility, as compared with a compound of which it is a derivative. For example, in some embodiments, the antioxidant is a water-soluble vitamin E derivative such as Trolox.

[00136] Those of skill in the art will appreciate that some of the compounds discussed herein may exhibit phenomena such as tautomerism and/or isomerism, e.g., stereoisomerism, or may exist in multiple crystalline forms. It should be understood that, in various embodiments, the invention encompasses use any tautomeric, isomeric, or polymorphic form of a compound having one or more of the uses described herein. For example, some compounds may contain one or more chiral centers and exist as two or more enantiomers. The invention encompasses embodiments in which a single enantiomer is used and embodiments in which multiple enantiomers are used, e.g., as a racemic mixture. In some embodiments, if the enantiomers have different effectiveness or potency, a more effective or more potent enantiomer is used. In some embodiments, a naturally occurring isomer is used. Some of the compounds may, in certain embodiments, bear one or more positive or negative charges and may have appropriate counter ions associated with them. For example, the compounds may be salts. The identity of the associated counter ion may be governed the synthesis and/or isolation methods by which the compounds are obtained. It will be understood that embodiments of the invention encompass compounds in association with any type of physiologically acceptable counter ion.

[00137] In some aspects, the invention provides culture medium suitable for deriving and/or maintaining ESCs, wherein the culture medium comprises at least one antioxidant. In some embodiments, the antioxidant is present at a concentration sufficient to preserve the XaXa status of XaXa ESCs when such ESCs are cultured under atmospheric 02 conditions. In some embodiments, the antioxidant is not an ingredient of a medium or substance known in the art to be useful for deriving or maintaining ESCs. For example, in some embodiments the antioxidant is a compound that is not an ingredient of DMEM/F12, serum, serum replacement, or other supplement used in the art for deriving or maintaining ESCs. In some embodiments, the compound is an ingredient of such medium or substance but, in some embodiments, is used at a higher concentration in the compositions or methods of the instant invention, wherein the concentration is sufficient to preserve the XaXa status of XaXa ESCs when such ESCs are cultured under atmospheric 02 conditions. In a non-limiting

embodiment, such concentration is at least 3-fold higher than the concentration present in DMEM/F12 (1 : 1 mixture). In some embodiments, the invention provides liquid culture medium comprising an antioxidant. In some embodiments, the invention provides at least some of the medium ingredients in dry form, e.g., as powders, to be dissolved. In some embodiments, the invention provides antioxidant preparations packaged in an amount suitable for a predetermined volume of culture medium, e.g., 450-500 mL or 900-1000 mL of medium, such that the resulting medium preserves the XaXa status of ESCs cultured in it under atmospheric 02 conditions.

[00138] In some embodiments of the invention, compound(s) are added to ESC derivation or maintenance medium at the time the medium is formulated (e.g., by a manufacturer or user) at around the same time as the majority of the ingredients are dissolved in water. In some embodiments, compound(s) are added shortly before the medium is used. In some embodiments, compound(s) are added directly to cell culture vessels. In some embodiments, the compound is added when media is changed and/or when cells are passaged. In some embodiments, compound(s) are added to the media between media changes or passagings. For example, compound(s) can be added daily or every other day.

[00139] In some embodiments, ESCs are derived and/or maintained in culture medium that has a reduced concentration, as compared with DMEM or DMEM/F12, of an uncomplexed metal (i.e., a metal not in a protein or other protective complex) that can contribute to production of free radicals and/or ROS. For example, the medium may have a reduced concentration of uncomplexed iron or copper as compared with DMEM or DMEM F12.

[00140] In some embodiments, at least two or three antioxidants are used in combination. For example, in some embodiments at least two antioxidants selected from the group consisting of vitamin C, vitamin E, lipoic acid, and morin hydrate are used. In some embodiments at least three antioxidants selected from the group consisting of vitamin C, vitamin E, lipoic acid, and morin hydrate are used. All combinations are encompassed. For example, in one embodiment, vitamin C, vitamin E, and lipoic acid are used in combination. In another embodiment vitamin C, vitamin E, lipoic acid, and morin hydrate are used in combination.

[00141] Compounds can be used at a range of concentrations. In some embodiments, a suitable concentration is between 1 nM and 1 mM, e.g., between 10 nM and 500 μΜ. For example, a number of antioxidants were shown to be effective at concentrations ranging from 1 μΜ (e.g., alpha tocopherol) to 100 μΜ (N-acetylcysteine) (see Table S3). Vitamin C, vitamin E, and lipoic acid were shown to be effective in combinations in which each compound was provided at 1 μΜ, 5 μΜ or 10 μΜ. It is anticipated that lower and higher concentrations would be effective. In some embodiments, concentrations may be varied by a factor of up to 100-fold lower or higher (i.e., between 0.01 and 100 times the listed concentration), e.g., up to 50-fold, 25-fold, 10-fold, 3-fold, or 2-fold lower or higher relative to the concentrations listed in Table S3. In some embodiments, vitamin C, vitamin E, lipoic acid, and/or morin hydrate is used at between 1 μΜ and 20 μΜ. In some embodiments, the sum of the molar concentrations of vitamin C, vitamin E, lipoic acid, and/or morin hydrate used is between 1 μΜ and 50 μΜ, e.g., between 1 μΜ and 5, 10, 20, 25, 30, 35, 40, or 45 μΜ. When two or more antioxidants are used, they can be used at the same concentration or at different concentrations. When used at different concentrations, they may be used at a selected ratio, which may be determined to be particularly effective. In some embodiments, multiple antioxidants are used, wherein at least one of the antioxidants is used at a lower concentration as compared with the concentration employed if the antioxidant is used as a single agent.

[00142] In some embodiments, antioxidant(s) are provided in amounts sufficient to individually or collectively provide at least about the same antioxidant capacity as 1 μΜ alpha tocopherol. In some embodiments, antioxidant(s) are provided in amounts sufficient to individually or collectively provide at least about the same antioxidant capacity as 5 μΜ lipoic acid. In some embodiments, antioxidant(s) are provided in amounts sufficient to individually or collectively provide at least about the same antioxidant capacity as a combination of 1 μΜ vitamin C, 1 μΜ vitamin E, and 1 μΜ lipoic acid. Antioxidant capacity can be measured as known in the art. For example, assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET) can be used. Many HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in reducing an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay using Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential "_a_ssay using a Cu(II) complex as an oxidant, and DPPH. In some embodiments, a TEAC assay is used. In some embodiments, an ORAC assay is used.

[00143] In some embodiments, a compound is provided as a substantially pure

preparation. For example, a preparation may consist of at least 80% of a particular compound by dry weight, e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more. In other embodiments, a preparation may consist of between 50% and 80% of a particular compound by dry weight. Any art-accepted method can be used to assess purity. For example, chromatography (e.g., gas chromatography, liquid chromatography), or mass spectrometry can be used.

[00144]

[00145] In some embodiments, an antioxidant or other compound used in the invention is physiologically acceptable in the context in which it is used, by which is meant that the compound is substantially non-toxic to the embryos, ESCs, and/or other desired cell type(s), e.g., at least at the concentration used. In some embodiments, a physiologically acceptable compound is also substantially non-toxic when used at higher concentrations (e.g., up to 10, 100, or 1000-fold higher). In some aspects, a substantially non-toxic compound does not cause a statistically significant decrease in embryo viability or grade and/or in cell viability and/or proliferation. Methods for assessing embryo viability or grade or cell viability or proliferation are known in the art. For example, the survival and/or proliferation of a cell or cell population can be determined using an assay selected from: a cell counting assay, a replication labeling assay, a cell membrane integrity assay, a cellular ATP-based viability assay, a mitochondrial reductase activity assay, a caspase activity assay, an Annexin V staining assay, a DNA content assay, a DNA degradation assay, and a nuclear fragmentation assay. Other exemplary assays include BrdU, EdU, or H3-Thymidine incorporation assays; DNA content assays using a nucleic acid dye, such as Hoechst Dye, DAPI, Actinomycin D, 7-aminoactinomycin D or propidium iodide; cellular metabolism assays such as AlamarBlue, MTT, XTT, and CellTitre Glo; Nuclear Fragmentation Assays; Cytoplasmic Histone Associated DNA Fragmentation Assay; PARP Cleavage Assay; TUNEL staining; and Annexin staining. In some embodiments, a compound does not cause significant reduction in pluripotency of ESCs at the concentration used. For example, in some embodiments the compound does not cause significant differentiation of ESCs at the concentration used. For example, the compound may not result in decreased expression of a pluripotency marker and/or may not result in increased expression of a differentiation-associated marker in a culture of ESCs exposed to the compound. In some embodiments, the invention provides medium or medium ingredients suitable for deriving or maintaining ESCs, wherein the medium or medium ingredient comes from a batch that has undergone quality control testing. For example, such quality control testing may include using a sample from the batch to derive and/or maintain ESCs. In some embodiments, ESCs of the invention, e.g., XaXa hESCs, are used for quality control testing. For example, a medium or medium ingredient could be tested for efficacy in preserving XaXa status under standard 02 conditions and/or to confirm that they do not contain undesired substances in amounts that could cause reduction in pluripotency and/or increased differentiation of ESCs. In some embodiments, the quality control tested ingredient comprises an antioxidant. [00146] Undifferentiated cells are often stored frozen - typically in the presence of a cryoprotectant such as glycerol under liquid nitrogen. Cryoprotectants are compounds that help protect biological material from freezing damage. Such compounds are often sugars or alcohols containing at least two hydroxyl groups. For example, ethylene glycol, propylene glycol, glycerol, DMSO (dimethyl sulfoxide), sucrose, and mixtures thereof are commonly used. As used herein, a "cryopreservation solution" is a composition comprising at least one cyroprotectant. In some embodiments, cells of the invention are frozen and stored in a cryopreservation solution. In some embodiments, cells of the invention are frozen and stored in the presence of one or more antioxidants. The invention provides cryopreservation solutions comprising an antioxidant. In some aspects, the antioxidant improves the recovery of XaXa hESCs after cryopreservation. In some aspects, the antioxidant improves the recovery of XaXa hESCs that retain the ability to undergo XCI upon differentiation.

[00147] III. Applications and Additional Embodiments

[00148] ES cells and cell lines of the invention have a wide variety of applications. In general, and without limitation, ESCs of the invention can be used for any purpose contemplated in the art for use of ESCs. Without wishing to be bound by any theory, ESCs of the invention may exhibit improved properties for use in such applications. Certain ESCs of the invention have a number of other uses, e.g., uses that relate at least in part to their X chromosome activation status.

[00149] In some aspects, ESCs of the invention are used for the study of events that occur in the early stages of embryonic development. For example, ESCs of the invention that have two active X chromosomes may be used to study XCI, e.g., to identify mechanisms and genes that regulate XCI. As another example, ESCs of the invention are useful in the study of mechanisms and genes that regulate cell differentiation.

[00150] In some embodiments, ESCs of the invention are used for screening purposes. For example, such cells can be used to screen for compounds or conditions that promote or inhibit differentiation, e.g., towards particular cell lineages or cell types. Such compounds of conditions can be used, e.g., to produce large quantities of differentiated cells of a desired cell lineage or cell type. In some embodiments, compounds or conditions identified using the inventive screening methods are used for modulating the differentiation of iPS cells. In other embodiments, ESCs of the invention are used in screens to identify compounds or conditions that promote or inhibit XCI. In still other embodiments, ESCs of the invention are used in screens to identify compounds or conditions that help ESCs survive freezing and thawing, help preserve ESCs in an XaXa state upon freezing and thawing, and/or help preserve the ability of of XaXa ESCs that have been frozen and thawed to properly undergo XCI when differentiated or shifted to atmospheric 02. "Freezing" for purposes of this invention, refers to cooling to temperatures at or below 0°C, such as below about -136°C, and typically to 77 K or -196 °C (the boiling point of liquid nitrogen). "Thawing" refers to returning previously frozen material to a temperature above 0°C, typically to about 37±1°C.

[00151] In some aspects, the invention provides methods for identifying a compound or condition that promotes or inhibits differentiation of ESCs, e.g., towards a particular cell lineage or cell type. In some embodiments, a method comprises contacting ESCs of the invention with a test compound and determining whether the presence of the test compound results in enhanced or inhibited differentiation (e.g., increased or decreased number of cells of a cell lineage or cell type of interest as compared with the number of such cells that would result in the absence of the test compound) relative to that which would occur if cells had not been contacted with the test compound. ESCs may be maintained in culture for, e.g., between 1 and 30 days, e.g., at least 3 days, at least 5 days, up to 10 days, up to 15 days, up to 30 days, etc., during which time they are contacted with the test compound for all or part of the time. In some embodiments the ESCs are cultured in the presence of one or more compounds or conditions known in the art to induce differentiation, in addition to being contacted with the test compound. For example, ESCs could be co-cultured with particular cell types, contacted with compounds known to induce differentiation, cultured in a vessel comprising a substrate that does not promote cell adhesion, cultured in suspension, etc. In some embodiments, the test compound is identified as a compound that that promotes or inhibits differentiation of ESCs if the number or percentage of cells that are differentiated to cells of a particular cell lineage or cell type of interest increases or decreases, respectively, by at least 1.25, 1.5, 2, 5, or 10-fold after said time period than would be present if the test compound had not been used. In some embodiments, a compound increases the efficiency of differentiation, results in a more homogeneous population of differentiated cells, and/or increases the speed at which differentiation occurs.

[00152] In some aspects, the invention provides methods for identifying a compound or condition that promotes or inhibits XCI. In some embodiments, a method comprises contacting ESCs of the invention with a test compound and determining whether the test compound results in enhanced or inhibited XCI (e.g., increased or decreased number of cells undergoing XCI upon differentiation or shift to atmospheric 02 conditions, as compared with the number of such cells that would undergo XCI upon differentiation or shift to atmospheric 02 conditions in the absence of the test compound) r relative to that which would occur if cells had not been contacted with the test compound. ESCs may be maintained in culture for, e.g., between 1 and 30 days, e.g., at least 3 days, at least 5 days, up to 10 days, up to 15 days, up to 30 days, etc., during which time they are contacted with the test compound for all or part of the time. In some embodiments the test compound is identified as a compound that that promotes or inhibits XCI if the number or percentage of ESCs that undergo XCI upon differentiation or shift to atmospheric 02 conditions increases or decreases, respectively, by at least 1.25, 1.5, 2, 5, or 10-fold after said time period relative to the number or percentage of ESCs that would undergo XCI upon differentiation or shift to atmospheric 02 conditions if the candidate compound had not been used.

[00153] In some aspects, the invention provides methods for identifying a compound or condition that helps preserve ESCs in an XaXa state upon freezing and thawing and/or helps preserves the ability of of XaXa ESCs that have been frozen and thawed to properly undergo XCI. In some embodiments, a method comprises contacting ESCs of the invention with a test compound, freezing the ESCs, thawing the ESCs, and assessing the X chromosome activation status of viable ESCs recovered after such thawing. In some embodiments, (i) cells are cultured in medium containing the test compound prior to being placed in a cryopreservation solution; (ii) the test compound is included in the cryopreservation solution; (iii) cells are contacted with the candidate compound during thawing; and/or (iv) cells are cultured in medium containing the test compound during or after thawing. If the use of the test compound results in enhanced recovery of XaXa ESCs, as compared with the number or percentage of XaXa ESCs that would be recovered if the test compound had not been used, the compound is identified as helpful to preserve ESCs in an XaXa state during freezing and/or thawing. In some embodiments, a compound increases the number or percentage of XaXa ESCs recovered by at least 1.25, 1.5, 2, 5, or 10-fold after said time period relative to the number or percentage of XaXa ESCs that would be recovered if the test compound had not been used. The invention further provides similar methods in which the ability of candidate compounds to enhance recovery of viable ESCs after freezing and thawing is assessed, e.g., in order to identify compounds that enhance recovery of such ESCs. The invention further provides similar methods in which the ability of test compounds to enhance recovery of viable ESCs that can properly undergo XCI upon differentiation is assessed, e.g., in order to identify compounds that enhance recovery of such ESCs. The invention further provides methods in which the ability of different freezing or thawing conditions (e.g., regimes of cooling and/or rinsing during warming) are assessed, e.g., to identify conditions that enhance recovery of viable XaXa ESCs after freezing and thawing and/or enhance recovery of viable XaXa ESCs that retain the ability to properly undergo XCI upon differentiation. For example, different rates of cooling or warming can be assessed.

[00154] A wide variety of compounds or combinations thereof can be used in aspects of the present invention, e.g., as test compounds in the inventive methods. For example, compounds may comprise e.g., polypeptides, peptides, small organic or inorganic molecules, polysaccharides, polynucleotides, oligonucleotides, peptide nucleic acids, or lipids.

"Polypeptide" is used interchangeably herein with "protein". Polypeptides can contain standard amino acids (which refers to the 20 L-amino acids that are most commonly found in naturally occurring proteins) and/or non-standard amino acids or amino acid analogs. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a moiety such as a carbohydrate group, a phosphate group, a fatty acid group, etc. "Peptide" is used herein to refer to a polypeptide containing 60 amino acids or less. "Polynucleotide" is used herein interchangeably with "nucleic acid" and encompasses single-stranded, double- stranded, and partially double-stranded molecules, double-stranded molecules with overhangs, etc. "Oligonucleotide" refers to a polynucleotide containing 60 nucleotides or less and encompasses antisense oligonucleotides, short interfering RNA (siRNA), and microRNA (miRNA). A polynucleotide can comprise standard nucleosides (which term refers to nucleosides that are most commonly found in DNA or RNA - adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), non-standard nucleosides,and/or nucleoside analog(s). Non-standard nucleosides can be naturally occurring nucleosides or may not be known to occur naturally. A non-standard nucleoside or nucleoside analog may differ from a standard nucleoside with regard to the base and/or sugar moiety. Variants of the sugar-phosphate backbone found in DNA or RNA can be used such as phosphorothioates, locked nucleic acids, or morpholinos. Modifications (e.g., nucleoside and/or backbone modifications), non-standard nucleotides, delivery vehicles and systems, etc., known in the art as being useful in the context of siRNA or antisense-based molecules for research or therapeutic purposes are contemplated for use in various embodiments of the invention. Such modifications may, e.g., increase stability, increase cell uptake, reduce clearance from the body, reduce toxicity, reduce off-target effects, or have other effects that may be desirable. "Small molecule" as used herein refers to a molecule having a molecular weight of not more than 1 ,500 Da, e.g., not more than 1000Da, e.g., not more than 500 Da. In some embodiments, the candidate compound is a small organic molecule comprising one or more functional groups that mediate structural interactions with proteins, e.g., hydrogen bonding. For example, a compound could comprise amine, carbonyl, hydroxyl or carboxyl group(s). In some embodiments a compound comprises one or more cyclic carbon or heterocyclic rings, e.g., an aromatic or polyaromatic ring substituted with one or more chemical functional groups and/or heteroatoms. In some embodiments, a small molecule has between 5 and 50 carbon atoms, e.g., between 7 and 30 carbons. Compounds can be contacted with cells by adding the compound to the culture medium. A range of concentrations can be used. Exemplary concentrations range from picomolar to millimolar, e.g., between 100 pM to 1 mM, e.g., between 10 nM and 500 μΜ. In some embodiments, a vector that encodes a candidate compound (an RNA or protein) is introduced into cells by an appropriate method and expressed therein to deliver a compound. For example, an expression vector that encodes a short hairpin RNA (shRNA) or microRNA (miRNA) precursor can be introduced into cells.

[00155] Compounds may be obtained from a wide variety of sources and can comprise compounds found in nature or compounds not known to occur in nature. Compounds can be synthesized or obtained from natural sources. For example, polypeptides may be produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis. Numerous techniques are available for the random and directed synthesis of a wide variety of organic compounds. In some

embodiments, candidate compounds are provided as mixtures of natural compounds in the form of bacterial, fungal, plant and animal extracts, fermentation broths, conditioned media, etc. In some embodiments, a library of compounds is screened. A library is typically a collection of compounds that can be presented or displayed such that the compounds can be conveniently used in a screening assay. Often, each compound has associated information stored, e.g., in a database, such as the chemical structure, purity, quantity, physiochemical characteristics of the compound and/or information regarding known or suspected biological or biochemical activity. In some embodiments, compounds or mixtures thereof are housed in individual wells (e.g., of microtiter plates), vessels, tubes, etc. Libraries include but are not limited to, for example, phage display libraries, peptide libraries, oligonucleotide libraries, siR A libraries, shRNA libraries, aptamer libraries, synthetic small molecule libraries, and natural compound libraries. Libraries could comprise multiple different compounds having a similar biological activity of interest. For example, libraries could comprise inhibitors of one or more enzymes or enzyme classes of interest. Exemplary compounds could be kinase inhibitors, phosphatase inhibitors, inhibitors of DNA or histone modifying enzymes (e.g., histone deacetylase inhibitors), etc. Methods for preparing libraries of molecules are well known in the art, and many libraries are available from commercial or non-commercial sources. In some embodiments, a library comprises between 1 ,000 and 1 ,000,000

compounds, or more, e.g., between 10,000 and 500,000 compounds. In many embodiments the candidate compound to be tested is a compound that is not present in ESC culture medium or cryopreservation solutions known in the art. In some embodiments a compound to be tested is a compound that is present in at least some ESC culture medium or

cryopreservation solutions known in the art but is used in a different, e.g., greater,

concentration in a method or composition of the present invention.

[00156] The invention encompasses the recognition that the X chromosome activation state of ESCs provides a biologically meaningful context in which to assess the ability of a compound to protect against cell stress. In some aspects, the invention relates to use of XaXa ESCs to assess a compound for potential therapeutic utility in a stress-related disease.

According to this aspect of the invention, a compound that at least in part preserves the XaXa state of ESCs when the ESCs are placed under a cell stress condition is a candidate compound for use in treating one or more such diseases. The cell stress condition could be, e.g., a non-physiological 02 condition and/or exposure to a cell stress inducer such as an oxidizing agent. The invention provides composition and methods for identifying and/or testing potential therapeutic agents for use in a stress-related disease. The invention provides a method of identifying a candidate ompound for a stress-related disease comprising steps of: (a) contacting a population of XaXa ESCs with a test compound; (b) subjecting the ESCs to a cell stress condition that promotes XCI; and (c) assessing the ability of the test compound to preserve the XaXa state of the ESCs, wherein a test compound that at least in part preserves the XaXa state of ESCs is a candidate compound for a stress-related disease. In some embodiments, a test compound at least in part preserves the XaXa state of ESCs if the percentage of ESCs that undergoes XCI when subjected to the cell stress condition is reduced, as compared with the percentage of ESCs that would undergo XCI when subjected to the cell stress condition if the test compound were not used. In some embodiments, the test compound does not significantly impair the ability of ESCs to properly undergo XCI upon differentiation. Thus a candidate compound in some embodiments does not simply block XCI entirely but rather preserves the XaXa state in the presence of cell stress, while not significantly inhibiting XCI from occurring under appropriate conditions. In some aspects, the invention further provides a method of identifying a candidate compound for a stress- related disease comprising steps of: (a) contacting a population of XaXa ESCs with a test compound; (b) subjecting the ESCs to a cell stress condition that promotes loss of ability of XaXa ESCs to undergo XCI upon differentiation; and (c) assessing the ability of the test compound to preserve the ability of XaXa ESCs to undergo XCI upon differentiation, wherein a compound that at least in part preserves the ability of XaXa ESCs to undergo XCI upon differentiation is a candidate compound for a stress-related disease. In some

embodiments, the ESCs have not been determined to have a genetic mutation, polymorphism, chromosomal abnormality, or other indicator of a genetically or epigenetically determined disease or syndrome. In other embodiments, the ESCs have been determined to have a genetic mutation, polymorphism, chromosomal abnormality, or other indicator of a genetically or epigenetically determined disease or syndrome. For example, the ESCs may ^" have been genetically modified to comprise a mutation, polymorphism, or chromosomal abnormality associated with a disease or may have been derived from an embryo that had a genetic mutation, polymorphism, or chromosomal abnormality. Such ESCs may have particular utility in the identification of candidate compounds for treating the disease and/or for studying disease pathogenesis. A candidate compound identified according to an inventive method may be further tested, e.g., in animal models or in humans. In some embodiments the trait or disease shows a simple Mendelian inheritance pattern. For example, the disease may be one that can be caused by a mutation in a single gene such as sickle-cell anemia or cystic fibrosis. In other embodiments the trait or disease may be affected by several genetic loci (and the environment). See, e.g., Mc usick, V.A.: Mendelian

Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology

Information, National Library of Medicine (Bethesda, MD), as of May 1 , 2010, World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/ and Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at http://omia.angis.org.au/contact.shtml. In some embodiments the trait or disease is inherited in a non-Mendelian fashion such as imprinting.

[00157] In some embodiments, human ES cells of the invention and/or cells or tissues derived therefrom, are used for toxicity screening, e.g., for embryotoxicity screening. In some aspects, a compound that causes primate, e.g., human, XaXa ESCs of the invention to undergo XCI while under physiological 02 is identified as potentially toxic, e.g, potentially deleterious to embryonic development. In some aspects, a compound that causes primate, e.g., human, XaXa ESCs of the invention to fail to undergo XCI following transfer to atmospheric 02 conditions and/or following transfer to differentiation conditions is identified as potentially toxic, e.g., potentially deleterious to embryonic development. In some aspects, a compound that causes primate, e.g., human, XaXa ESCs of the invention to fail to maintain XIST expression following transfer to atmospheric 02 conditions and/or following transfer to differentiation conditions is identified as potentially toxic, e.g., potentially deleterious to embryonic development. In some embodiments, a compound that causes primate, e.g., human, ES cells of the invention to fail to differentiate into cells of the three germ layers and/or to fail to form normal embryoid bodies (EBs) when placed under conditions in which such differentiation would otherwise be expected is identified as potentially toxic, e.g, potentially deleterious to embryonic development. As known in the art, EBs are aggregates of cells that typically begin as irregular clumps in cultures of embryonic stem cells that exhibit differentiated tissue types within their structure. The invention provides EBs obtained from ESCs derived under physiological 02 conditions. EB formation can be promoted, e.g., by culturing ESCs in suspension, e.g., in hanging drops, or in culture vessels that do not favor cell adhesion and attachment. See Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng.

103(5):389— 398, 2007, for a review describing several common EB culture methods. In some embodiments, EBs are obtained under physiological 02 conditions. The inventive toxicity screening methods may be used, for example, to test naturally occurring and/or synthetic compounds, compounds of demonstrated or potential therapeutic utility, compounds that may be released into the environment or to which humans or animals may be exposed, e.g., as ingredients of pesticides, herbicides, cleaning agents, foods, cosmetics, etc., through use in manufacturing or other industrial processes, or as wastes arising from such processes.

[00158] ESCs of the invention may be induced to differentiate into desired cell types. Such differentiated cells are an aspect of the invention. For example, the ESCs may be induced to differentiate into hematopoietic stem cells, neural lineage cells, striated muscle cells, cardiac muscle cells, liver cells, pancreatic cells, cartilage cells, epithelial cells, urinary tract cells, ocular cells (e.g., retinal cells, limbal epithelial stem cells), vascular cells etc., by culturing such cells in differentiation medium and under conditions which provide for cell differentiation. Cell types of interest include, without limitation, keratinocytes, pigmented retinal epithelium, neural crest cells, motor neurons, dopaminergic neurons, hepatic progenitors, pancreatic islet-like cells (e.g., insulin-secreting beta-like cells), and

mesenchymal stem cells. In some embodiments, differentiation is performed under physiological O₂ conditions or under conditions that mimic physiological 02 conditions. For example, ESCs may be differentiated in culture medium comprising an antioxidant, e.g., in an amount sufficient to preserve the XaXa state of XaXa ESCs. In some embodiments, the invention provides a differentiated cell, wherein the cell is descended from an ESC derived and maintained under physiological 02 conditions or conditions that mimic physiological 02 conditions, and wherein differentiation to obtain the cell is performed under physiological 02 conditions or conditions that mimic physiological 02 conditions. In other embodiments, differentiation is performed under non-physiological O₂ conditions, e.g., atmospheric 02 conditions. In some embodiments, ESCs are differentiated to the endodermal,

mesendodermal, or neuroectoderm lineage. In some embodiments a cell type of interest is a stem cell. A stem cell is capable of self-renewal and of differentiating to at least one more mature cell type. In some embodiments, a stem cell is a multipotent stem cell. A multipotent stem cell can give rise to cells of multiple different types but has less potential than a pluripotent cell. Exemplary multipotent stem cells include mesenchymal stem cells, neural stem cells, hematopoietic stem cells and more restricted hematopoietic cells such as myeloid or lymphoid stem cells, endothelial stem cells, etc. Cell types of interest can be identified, e.g., by cell surface markers, expression of reporter genes, gene expression profile, and/or characteristic morphology. If desired, a cell population can be enriched for cell type(s) of interest and/or further cultured to obtain more mature cell type(s). In some embodiments, enrichment comprises selecting cells that express one or more markers associated with the desired cell type(s) and/or selecting cells that do not express one or more markers associated with pluripotency. In some embodiments, enrichment comprises removing at least some cells that express one or more markers associated with pluripotency from the cell population. In some embodiments, the invention provides a differentiated cell population obtained from ESCs of the invention, wherein the cell population is substantially free of pluripotent cells. In some embodiments, no more than 5%, 2%, 1 %, 0.5%, 0.1% or 0.05% of the cells express a marker associated with pluripotency. In some embodiments, expression of said marker is not significantly greater than a reference level, e.g., a background or control level.

[00159] Medium and methods which result in the differentiation of ES cells are known in the art as are suitable culturing conditions. The differentiation of hESCs into a variety of cell and tissue types often involves the formation of EBs. Differentiation along lineages of interest can be promoted by a variety of different compounds such as polypeptides, nucleic acids, and small molecules. Exemplary compounds include growth factors, morphogenetic factors, and small cell-permeable molecules such as steroids (e.g, dexamethasone), vitamins (e.g., vitamin C), sodium pyruvate, thyroid hormones, prostaglandins, dibutryl cAMP, concavalin A, vanadate, and retinoic acids. For example, e.g., bone morphogenetic proteins such as BMP-2 are useful to promote chondrogenic differentiation. Mechanical factors (e.g., mechanical properties of a scaffold or culture substrate, application of forces) can also promote differentiation along particular pathways. By way of example, methods useful for generating ES cell-derived dopaminergic neurons are described in Kriks S, Studer L., Adv Exp Med Biol., 651 : 101-1 1 , 2009; methods useful for directing chondrogenic differentiation of ES cells using various growth factors such as BMP2 and TGF i are described in Toh, WS, et al., "Differentiation of Human Embryonic Stem Cells Toward the Chondrogenic Lineage", in Stem Cell Assays, Methods in Molecular Biology, Volume 407,^'pp. 333-359, Humana Press, Totowa, NJ, 2007; methods useful for directing chondrogenic differentiation of ES cells using various growth factors such as BMP2 and TGFpi are described in Toh, WS, et al., "Differentiation of Human Embryonic Stem Cells Toward the Chondrogenic Lineage", in Stem Cell Assays, Methods in Molecular Biology, Volume 407, pp. 333-359, Humana Press, Totowa, NJ, 2007; methods useful for generating cardiomyocytes are described in Cao F, et al., "Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes" PLoS One. ;3(10):e3474, 2008. These references are merely exemplary of reported methods for obtaining differentiated cells from ES cells. In some aspects, the invention provides a composition comprising: (a) one or more ESCs derived under or substantially under physiological 02 conditions; and (b) one or more material(s) that promotes differentiation of the ESC(s) to one or more cell type(s) of interest. The material(s) could be, e.g., compound(s), a substrate, or cells. In other aspects, the invention provides a method of generating a cell type of interest comprising: (a) providing one or more ESCs derived under or substantially under physiological 02 conditions; and (b) culturing the one or more ESC of step (a) under conditions that promote differentiation of said cell(s) to one or more cell types of interest. In some embodiments, the conditions comprise culturing the cell(s) in culture medium comprising one or more compound(s) that promote

differentiation to a desired cell lineage or cell type. Furthermore, the invention encompasses use of ESCs of the invention to screen test compounds (e.g., test compounds such as those described above), to identify compounds that promote differentiation of pluripotent cells (e.g., ESCs or iPS cells) to one or more desired cell types.

[00160] Differentiated cells of the invention, e.g., differentiated human cells, have a variety of uses. In some embodiments, such cells are used for therapeutic purposes. For example, neural lineage cells could be used to treat, prevent, or stabilize a neurological disease such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS, lysosomal storage diseases, multiple sclerosis, or a spinal cord injury. Differentiated cells that produce a hormone, such as a growth factor, thyroid hormone, thyroid-stimulating hormone, parathyroid hormone, steroid, serotonin, epinephrine, or norepinephrine may be administered to a mammal for the treatment or prevention of endocrine conditions.

Differentiated cells may be administered to repair damage to the lining of a body cavity or organ, such as a lung, gut, exocrine gland, or urogenital tract or to treat damage or deficiency of cells in an organ or tissue such as the bladder, bone, bone marrow, brain, cartilage, esophagus, eye, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, muscle, ovaries, pancreas, prostate, skin, spinal cord, spleen, stomach, tendon, testes, thymus, thyroid, trachea, ureter, urethra, or uterus. Differentiated cells could be used in tissue engineering, e.g., the construction of a replacement organ or tissue ex vivo. For example, such cells could be combined with a suitable scaffold, which is optionally three-dimensional and/or biodegradable. Optionally, the cells are allowed to proliferate and possibly further differentiate ex vivo. Scaffolds could be comprised of a wide variety of materials, including both naturally occurring and artificial materials. See, e.g., Lanza, R., et al. (eds.), Principles of Tissue Engineering, 3^rd ed., Academic Press, 2007. The replacement organ, tissue, or portion thereof is transplanted into a recipient in need thereof. In some embodiments, differentiated cells or organs or tissues comprising them are introduced into a non-human animal that serves as a model of a disease. The term "disease" as used herein, encompasses, in various embodiments, art-recognized diseases, disorders, syndromes, injuries, impairments of health or conditions of abnormal functioning, e.g., for which medical/surgical treatment would be desirable. The non-human animal may then be assessed, e.g., to evaluate the effects of the introduced cells, organs, or tissues in the model, thus providing means to assess therapeutic potential. As used herein, "treat", "treating", "therapy" and similar terms can include amelioration (e.g., reducing one or more symptoms of a disorder), cure, and/or maintenance of a cure (i.e., the prevention or delay of recurrence) of a disorder, or preventing a disorder from manifesting as severely as would be expected in the absence of treatment. Treatment after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder, and/or at least some of its associated symptoms, to prevent it from becoming more severe, to slow the rate of progression, or to prevent the disorder from recurring once it has been initially eliminated. Treatment can be prophylactic, e.g., administered to a subject that has not been diagnosed with the disorder, e.g., a subject with a significant risk of developing the disorder. For example, the subject may have a mutation associated with developing the disorder. In some embodiments, e.g., in the case of a disorder diagnosed prior to birth, treatment can comprise administering a compound to a subject's mother. In some

embodiments, a method of the invention comprises diagnosing a subject as having or being at risk of developing a disease, or providing such a subject, and treating the subject. In some embodiments, a subject diagnosed or treated according to the instant invention is a human. In some embodiments a subject is a non-human mammal, e.g., any of the mammals mentioned herein.Differentiated cells of the invention can also be used for screening or other testing purposes, e.g., to identify compounds of use for treating diseases, to assess the effects of a compound on such cells (e.g., to assess potential toxicity or explore mechanism of action) or to study a cell biological process of interest. For example, neural cells could be used to study neurotransmitter synthesis, release, or uptake and/or to identify compounds that modulate, e.g., promote or inhibit, such processes. Hepatocytes could be used in the study of drug metabolism and/or drug interactions. As another example, cardiomyocytes can be used in study of processes such as action potential generation, repolarization, excitation-contraction coupling or calcium flux and/or to identify compounds that modulate such processes.

Compounds so identified could be used in research or in treatment of diseases in which such modulation would be beneficial. The cells could be used in preclinical toxicology studies. For example, they could be used to assess potential cardiotoxicity, hepatoxicity,

neurotoxicity, drug interactions, etc. In still other embodiments, differentiated cells of the invention could be used in screens to identify compounds useful to direct endogenous cells to participate in the repair or regeneration of damaged tissues in vivo.

[00161] In some aspects, the invention provides methods of producing non-human mammals using non-human ESCs of the invention. In some aspects, the invention provides non-human mammals, e.g., transgenic non-human mammals, generated using ESCs of the invention. The non-human mammals can be genetically modified or non-genetically modified. In some embodiments the ESC or iPS cell has a mutation or polymorphism associated with a trait or disease that has a genetic component. In some embodiments, non- human mammals are produced using methods known in the art for producing non-human mammals from non-human ES cells.

[00162] In some embodiments, the non-human mammal serves as a model for a human disease. Such models are useful, e.g., for studying physiological processes or disease pathogenesis, testing the effect of a compound on the mammal, e.g., testing potential treatments, etc. In other embodiments, ESCs, iPS cells, or ESC-like cells could be used to generate farm animals (e.g., cows, pigs, sheep, goats, horses), e.g., farm animals with desired traits. Examples of such traits could be, e.g., reduced susceptibility to disease, increased size, increased milk production, etc.

[00163] In some embodiments, non-human mammals are useful for research on apoptosis, autoimmune disease, cancer, cardiovascular disease, cell biology, dermatology, development, diabetes and/or obesity, endocrine deficiency, hearing (or hearing loss), hematological research, immunology, inflammation, musculoskeletal disorders, neurobiology,

neurodenerative disease, metabolism, vision (or vision loss), reproductive biology, or infectious disease. Research can include, e.g., identification of targets for development of therapeutic agents, testing potential therapeutic agents, toxicity testing, etc.

[00164] [00165] In some embodiments, a non-human mammal of the invention is used as a model for a disease for which a pharmacological treatment is sought. In one embodiment, a method of identifying a compound to be administered to treat a disease in a mammal comprises providing a non-human mammal generated using an ESC of the invention; administering a test compound to the non-human mammal, wherein the test compound is to be assessed for its effectiveness in treating the disease; and assessing the ability of the compound to treat the disease. If the test compound reduces the extent to which the disease is present or progresses or causes the disease to reverse (partially or totally), or reduces one or more symptoms or signs of the disease, the test compound is identified as a compound to be administered to treat the disease.

[00166] IV. Kits

[00167] The invention provides a variety of kits. A kit can contain any of the cells or compounds described herein or combinations thereof. In some aspects, the invention provides a kit containing cells of an ESC line of the invention. The cells can be provided frozen. In some embodiments, the kit further comprises at least one item selected from the group consisting of (i) instructions for thawing, culturing, and/or characterizing the ESCs; (ii) reagent(s) useful for characterizing the ESCs. Such reagent could be, e.g., antibody(ies) for detecting a cell marker or probe(s) (e.g., for performing FISH). In some embodiments, the kit comprises reagents for assessing the X chromosome activation state of ESCs.

[00168] The invention further provides a kit comprising: ingredients for a cell culture medium suitable for deriving or culturing human ES cells, wherein the ingredients include at least one compound in an amount sufficient to mimic the effect of physiological 02 conditions on XaXa ESCs, and wherein the kit optionally comprises at least one item selected from the group consisting of: (i) instructions for preparing the medium; (ii) instructions for deriving or culturing human ES cells; (iii) serum replacement; (iv) albumin; (v) at least one protein or small molecule useful for deriving or culturing human ES cells, wherein the protein or small molecule activates or inhibits a signal transduction pathway; (vi) at least one reagent useful for characterizing human ES cells; and (vii) ESCs, which are optionally ESCs of the invention. In some embodiments, the ingredients comprise the ingredients present in DMEM or DMEM:F12. In some embodiments, the at least one compound that mimics the effect of physiological 02 conditions is an antioxidant, e.g., an antioxidant selected from the group consisting of: vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof. In some embodiments, medium is provided as a liquid, wherein at least some ingredients that can be stored for prolonged periods in liquid are dissolved therein.

Ingredients that are unstable or perishable or otherwise not suitable for long-term storage in liquid or are incompatible with each other can be provided in one or more separate containers. For example, one or more separate containers could contain supplements to be added shortly before the medium is used. In some embodiments, at least some of the ingredients are provided in dry form. For example, the ingredients can be provided in appropriate amounts so that the user can dissolve them in an appropriate volume of sterile water, e.g., distilled or deionized water. In some embodiments, at least some of the components of the kit are quality tested, e.g., using an ESC line of the invention.

Examples

Example 1 : Derivation of Pluripotent hES Cells in a Physiological Oxygen Environment

[00169] A total of 20 IVF-derived 8-cell embryos were thawed, cultured in 5% O₂, and allowed to develop to the blastocyst stage prior to hESC derivation. An additional 5 embryos were used for control derivations at 20% O₂ (Table SI). We established 3 hES lines from blastocysts grown in 5% O₂ (designated WIBR1 , 2, and 3; Figure S I A) as well as one line from 5 blastocysts in 20% O₂ (not shown). Immediately after stable establishment of hESCs, each line was split in half at the first passage, with one half remaining at 5% O₂ and the second half shifted to culture in 20% O₂. The resulting 3 pairs of genetically identical hES cell lines were cultured for prolonged periods of time (>18 months, Table 1). We will refer to these cell lines by their number followed by the oxygen concentration in which they were cultured, eg. WIBR1^5% or WIBR1^20%.

[00170] All three pairs of cell lines maintained genetic stability during the culture period, exhibiting normal karyotypes after chronic exposure to either physiological or atmospheric O₂ levels (Figure S2). We examined the expression of pluripotency markers in hESC colonies in both physiological and atmospheric O₂ conditions and observed no qualitative differences in the staining patterns of hES cell surface markers SSEA4, TRA-1-81 and TRA1-60, nor in the core pluripotency transcription factors OCT4, SOX2, and NANOG (Figure SIB). Consistent with the expression of these canonical pluripotency markers, all pairs of hESC lines were able to form complex teratomas containing tissues derived from the three germ layers upon injection into immunocompromised mice (Figure S3) and induced expression of transcripts associated with lineage commitment during embryoid body formation (Figure S4), demonstrating their pluripotency.

Example 2: Gene Expression Changes Induced by Exposure to Atmospheric Oxygen

[00171] In order to assess changes in gene expression induced by hyperoxic culture conditions, we performed microarray profiling on RNA purified from mechanically isolated, undifferentiated hESC colonies from lines WIBR1 , 2, and 3 after acute (72 hrs) or chronic exposure to 20% O₂ and compared the transcriptional profiles to cultures maintained in 5% O₂. Acute exposure to 20% O₂ induced a dramatic change in global gene expression and resulted in a marked downregulation of genes associated with hypoxia and the HIF pathway (Figure 1 A). Many of these changes were not maintained and approached baseline (5%) levels after chronic exposure to 20% O₂ (Figure 1A). Consistent with these observations, hierarchical clustering revealed that under either chronic 20% or 5%» O₂ culture conditions gene expression profiles tended to cluster hESC lines based on genetic background rather than O₂ culture conditions (Figure I B). Ultimately, we identified 198 genes whose change in expression after acute exposure to 20% O₂ was maintained during chronic culture (Figure S5A and Table S2. Gene set enrichment analysis across the three cell lines identified the upregulation of gene sets associated with mitochondrial activity and mRNA processing after acute exposure to atmospheric O₂ (Figure S5B). Gene sets associated with hypoxia such as the HIF, glycolysis, and gluconeogenesis pathways were identified as being downregulated in response to both acute and chronic atmospheric O₂ exposure (Figure S5C). Quantitative RT- PCR analysis on a number of embryonic stem cell and lineage-specific transcripts revealed no significant changes in the expression of the core pluripotency transcription factors OCT4, SOX2, and NANOG, whereas induction of neurectodermal, trophoblast, mesoderm, extraembryonic endoderm, and visceral endoderm genes was observed in hESC colonies after chronic exposure to 20% O₂ (Figure S5D). This suggests that the exposure to atmospheric hyperoxia does not significantly compromise pluripotency but may promote differentiation in a fraction of the cells.

Example 3: Predisposition for Spontaneous Differentiation

[00172] While the previous Example indicated that chronic exposure to 20% O₂ does not compromise expression of core pluripotency network genes in RNA derived from mechanically isolated hESC colonies, it does not rule out the possibility that atmospheric oxygen increases the susceptibility of hESCs to undergo spontaneous differentiation under suboptimal culture conditions. To address this possibility, we cultured hESC colonies on mouse embryonic fibroblasts (MEFs) constitutively expressing green fluorescent protein (GFP) for 8 days without passaging, allowing colonies to overgrow thereby triggering spontaneous differentiation. We then assessed the proportion of all human cells in the culture expressing SSEA4 and OCT4 using flow cytometric analysis, gating out the GFP-positive MEFs. Under these conditions, we observed a reduction in the fraction of OCT4 positive cells in WIBRl and WIBR2 after acute (72hr) as well as chronic exposure to 20% O₂ (Figure 1 C). In contrast WIBR3 remained largely unaffected, with over 90% of cells maintaining OCT4 immunoreactivity under all culture conditions. SSEA4 expression only significantly decreased in response to atmospheric oxygen in the male line WIBRl (Figure 1C). This finding highlights the variability of hESC lines in their response to atmospheric oxygen levels and may provide an explanation for the conflicting reports in the literature (Chen et al., 2009; Ezashi et al., 2005; Forsyth et al., 2008; Prasad et al., 2009). Taken together, our analysis indicates that while atmospheric oxygen exposure allows for a greater degree of spontaneous differentiation, the majority of cells in the cultures are able to actively maintain a pluripotent state.

Example 4: Imprinting in hESC Lines Derived and Cultured under Atmospheric or

Physiological Oxygen Conditions

[00173] To evaluate the consequences of long-term exposure to 20% O₂ on the epigenetic stability of the cells we assessed the fidelity of imprinting of several known imprinted genes using Sequenom matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry analysis of bisulfite modified DNA. This highly sensitive technique allows for the quantification of methylation at each individual CpG residue across a gene. We observed no significant differences in DNA methylation between 5% or 20% O₂ cultures across the three hESC lines at a number of imprinted loci including DLX5, H19, KCNQ1 , NDN, PLAG1 , SLC22A18, and SNURF (Data not shown). One exception was observed at PEG3 where chronic exposure to atmospheric oxygen was associated with increased CpG methylation. We conclude that the DNA methylation status at numerous imprinted loci remains stable in hESC lines cultured in both atmospheric and physiological oxygen environments.

Example 5: X Chromosome Inactivation

[00174] We next assessed the status of X chromosome inactivation in the two female lines (WIBR2 and 3). This is of particular interest since human ES cells are similar to murine EpiSCs and unlike murine ES cells (Brons et al., 2007; Tesar et al., 2007) in that they have invariably undergone XCI in most or all cells within a culture (Hall et al., 2008; Shen et al., 2008; Silva et al., 2008), consistent with the hypothesis that hES cells may not be

developmentally equivalent to ICM-derived murine ES cells but rather may correspond to the more mature epiblast-derived EpiSCs (which have a silenced X chromosome, (Guo et al., 2009; Nichols and Smith, 2009). Alternatively, the cells of the human ICM, in contrast to the mouse, may have already undergone X inactivation (van den Berg et al., 2009).

[00175] To assess the status of XCI in hESCs, we used fluorescent in situ hybridization (FISH) to visualize the non-coding XIST RNA. Under physiological O₂ concentrations, both female lines WIBR2 and WIBR3 exhibited no XIST positive cells and no detectible XIST gene expression as measured by quantitative RT-PCR (Figure 2A, 2B, S6A, and S6B). Since established hESC lines are known to silence XIST after undergoing XCI, we tested the ability of WIBR2 and WIBR3 to activate XIST upon differentiation. In contrast to established hESCs cultured under atmospheric oxygen, both female hESC lines maintained under physiological oxygen, similar to murine ES cells, activated XIST gene expression and formed an XIST cloud on the Xi upon differentiation, indicating that these cells exist in a XaXa state when maintained under 5% oxygen. We further confirmed the XaXa status of these cells by performing FISH for X-linked transcripts revealing two foci in cells cultured at 5% oxygen (Figure S7).

[00176] In contrast to cell lines maintained at 5% O₂, WIBR3^20% expressed high levels of XIST and had XIST clouds coating the Xi in 67% of cells, with the remaining cells undergoing XCI upon differentiation (Figure 2A and 2B). X chromosome DNA FISH confirmed that these cells maintained two X chromosomes in both 5% and 20% oxygen and revealed a full coating of the X chromosome territory by XIST RNA at 20% oxygen (Figure S8). Furthermore, FISH for X-linked transcripts showed a single focus in cells cultured at 20% oxygen, consistent with these cells having one inactivated X chromosome (Figure S7). This demonstrates that the cells cultured in 20% O₂ had undergone precocious XCI as has previously been observed in established hESC lines. WIBR2^20% did not exhibit XIST expression or XIST clouds and did not activate XIST upon differentiation (Figure S6A and S6B), suggesting that this line is defective in X chromosome inactivation as has been observed previously in some established hESC lines (Hall et al., 2008; Shen et al., 2008; Silva et al., 2008). To correlate the X chromosome inactivation status and XIST expression with CpG methylation of the XIST promoter, we performed Sequenom matrix-assisted MALDI-TOF mass spectrometry analysis of bisulfite modified DNA containing the two CpG islands around the XIST transcriptional start site. The XIST promoter in the male line WIBRl as well as in the female lines WIBR2^5% and WIBR3^5% exhibited over 90% methylation (Figure 2C, 2D, and Figure S6C). The high level of CpG methylation at the XIST promoter in undifferentiated female hESC lines is consistent with the lack of observed XIST expression. In contrast, female murine ES cells have only partially methylated Xist regulatory regions (approximately 50% on each of the two alleles; (Nesterova et al., 2008; Sun et al., 2006).

[00177] Under atmospheric O₂, line WIBR3^20% underwent a reduction of CpG methylation to 50%-60% (Figure 2D) consistent with one XIST allele having been activated. Bisulfite conversion and sequencing of individual clones revealed that these promoter regions were either completely unmethylated or heavily methylated, supporting the conclusion that this cell line has one active and one inactive XIST allele (Figure 2E). Line WIBR2 °, which exhibited no XIST foci and was unable to activate XIST expression upon differentiation, had an intermediate level of CpG methylation, possibly reflecting earlier XIST activation and XCI which was subsequently followed by XIST gene silencing (Figure S6C). To address this possibility, we performed FISH at the earliest passage of WIBR^20% from which enough material was available. We found that WIBR2^20% contained XIST foci in nearly all cells confirming that XCI had occurred during very early stages of culture and was followed by silencing of XIST expression and loss of XIST clouds on the Xi during the prolonged culture period (Figure S6E).

Example 6: Allele-Specific Expression of X-linked Genes

[00178] X-linked single nucleotide polymorphisms (SNPs) were identified in WIBR2 and WIBR3 to directly measure allele-specific gene expression using SNP-Chip and Sequenom dye termination mass-spectometry based expression analyses. Initial SNP-Chip analysis of WIBR2 and WIBR3 revealed biallelic expression of nearly all of the X-linked SNPs in cells cultured under 5% O₂ consistent with these lines carrying two active X chromosomes (XaXa) (Figure 3A and S9A). Under atmospheric oxygen concentrations, however, WIBR2^20% exhibited monoallelic expression at 31% of the SNPs (14/45). This is further evidence that this line, while lacking persistent XIST foci, underwent XCI (Figure S9A). WIBR3^20% exhibited monoallelic expression at 73% of X-linked SNPs (with SNPs exhibiting biallelic expression lying primarily in the pseudoautosomal region known to escape XCI, Figure 3B), suggesting that this line had undergone X chromosome inactivation producing a clonal population of cells with the same X chromosome being active in all cells.

[00179] Further quantification of allele-specific gene expression using Sequenom dye- termination RT-PCR followed by mass-spectrometry based analysis demonstrated that both WIBR2^5% and WIBR3^5% expressed equal levels of transcript from both X-chromosomes (Figure 3C and S9B). Under atmospheric oxygen conditions line WIBR3^20% (XaXi and XIST positive) exhibited nearly complete monoallelic expression of the X-linked genes examined (Figure 3D). In contrast, WIBR2^20% (XaXi, but lacking XIST foci) expressed the majority of X-linked transcripts from a single allele. However, significant transcripts were detected from the putative Xi suggesting that the lack of XIST expression and loss of XIST clouds may have resulted in a partial derepression of silenced genes on the Xi (Figure S9C and S9D).

[00180] Monoallelic expression of X-linked genes as observed in most established hESC cultures could be explained by precocious XCI induced under 20% O₂ culture conditions followed by clonal outgrowth of some cells, perhaps those cells best adapted to the suboptimal growth conditions. We directly tested whether human ES cells undergo random XCI by differentiating WIBR3^5% in vitro for two weeks, a period of time long enough for cells to undergo XCI (Figure 2A), but short enough that there would not be sufficient cell division for cells to undergo clonal selection. We were readily able to detect transcripts originating from both X chromosomes after differentiation, confirming that human ES cells undergo random X chromosome inactivation in a manner analogous to what has been observed in the murine system (Figure 3E). This finding is consistent with the notion that the monoallelic expression of X-linked genes observed in long-term cultures of XaXi hESC cultures is a result of clonal selection.

[00181] Example 7: X Inactivation in Response to Oxidative Stress

[00182] To determine whether the X chromosome inactivation in female hESCs cultured in atmospheric oxygen was reversible and whether exposure to atmospheric oxygen was sufficient to induce silencing of the X chromosome, we performed RNA FISH and qRT-PCR for XIST in cultures of WIBR2 and WIBR3 that were switched from either 5% to 20% O₂ or 20% to 5% O₂ for two weeks. Both WIBR2 and WIBR3 cells maintained in 5% O₂ began to upregulate XIST transcript levels and initiated the formation of small XIST foci upon exposure to atmospheric oxygen (Figure 2B, 2F and S6D). There were often two small XIST foci formed after transfer from 5% to 20% O₂, indicating that these cells had not yet committed to permanent silencing of one particular X chromosome (Panning et al., 1997). Conversely, return of cells maintained in atmospheric oxygen to 5% O₂ could not reverse the established Xi in WIBR3^20% (Figure 2F) and had no effect on WIBR2^20% (Figure S6D).

Allele-specific expression analysis of,WIBR3^5% after two weeks exposure to atmospheric oxygen showed biallelic expression of X-linked transcripts further supporting the notion that chromosome choice for XCI is random in human cells. These findings demonstrate that exposure to atmospheric oxygen alone is sufficient to drive irreversible X chromosome inactivation in human ES cells.

[00183] It is worth noting here that in addition to atmospheric oxygen exposure, we observed promiscuous XCI in response to cellular stress induced by harsh freeze-thaw cycles in which few hESC colonies were recovered. One particular thawing of WIBR2^5% that resulted in low cell viability (only 2-3 hESC colonies recovered) exhibited XCI. Here we observed XIST foci on almost all cells of the culture, XIST expression levels equivalent to those seen upon differentiation, and a demethylation of the XIST promoter despite the maintenance of these cells in 5% O₂ (Figure 4A-C). This finding prompted us to examine whether a general cellular stress response was sufficient to induce XIST activity. To this end, we cultured the XaXa hES cell line WIBR2^5% in the presence of a variety of cellular stress- inducing compounds while maintaining the cultures at 5% oxygen. We found that proteosome inhibition, HSP90 inhibition, gamma-glutamylcysteine synthetase inhibition, and organic peroxide treatment were all capable of activating XIST gene expression under 5% oxygen (Figure 4D and Table S3). Thus, the XaXa state of hES cells is precarious and prone to X chromosome inactivation by cellular stress.

[00184] Example 8: Antioxidant-Mediated Protection Against XCI

[00185] Given our findings that induction of oxidative stress using organic peroxides is sufficient to activate XIST under 5% oxygen, and that exposure to 20% oxygen is also sufficient to drive XCI, we reasoned that addition of antioxidants to the culture media might prevent XCI induced upon moving hES cells from 5% to 20% oxygen. We therefore added a number of antioxidants as well as a HIFla stabilizer (since we observed consistent suppression of the HIF pathway upon exposure to 20% oxygen, Figures 1 A and S5) to WIBR2 cells cultured in 5% oxygen prior to moving them to 20% oxygen (Table S3). We also included several histone deacetylase inhibitors in this panel of compounds because HDAC inhibition has previously been shown to suppress XIST expression in H9 hESCs (Ware et al., 2009) (Table S3). WIBR2 cells were subsequently cultured in 20% oxygen with daily addition of these compounds to the culture media, and after 24 days undifferentiated colonies were mechanically harvested and XIST expression was analyzed by FISH and qRT- PCR. Remarkably, all of these compounds were able to suppress XIST expression when compared to 20% oxygen controls, with several antioxidant-cultured cells exhibiting undetectable levels of XIST expression indistinguishable from the parental 5% oxygen control cultures (Figure 4E). We confirmed the presence of XIST clouds in untreated 20% oxygen cultures and observed no XIST foci in the antioxidant treated cultures (Figure 4F), demonstrating that inhibition of oxidative stress is sufficient to protect hES cells from precocious XCI after exposure to atmospheric oxygen.

[00186]

Example 9: Genome-Wide Analysis of Histone Modifications

[00187] To correlate the activity of the X-chromosome with the chromatin state in hESC lines cultured in atmospheric or physiological oxygen concentrations, we performed genome- wide location analysis using chromatin immunoprecipitation for histone modifications that mark active (H3K4-me3) and repressed (H3 27-me3) genes followed by sequencing the immunoprecipitated DNA fragments (ChlP-Seq) on the three pairs of hES cell lines. The distribution of these histone modifications on autosomes was grossly unaffected by changes in oxygen concentration (Figures 5 A and S10). We did, however, observe a dramatic accumulation of H3K27 trimethylation on the putative Xi of the female cell lines WIBR2^20% and WIBR3^20% (Figure 5B). This accumulation was not observed in the female hESC lines maintained under physiological oxygen or in the male cell line WIBR1 in either oxygen environment. This is consistent with the observed monoallelic expression originating from the X chromosome of female hESCs maintained in 20% O₂, and further supports the conclusion that these cells have undergone XCI. Interestingly, the previous observation that WIBR2 ^20% had lost the XIST coating on the Xi demonstrates that the repressive histone modifications are sufficient to maintain a level of gene silencing in the absence of XIST in human ES cells, and that the continued presence of XIST is not required for maintaining the repressive H3K27me3 modification along the Xi as has previously been suggested by immunofluorescence analysis (Shen et al., 2008; Zhang et al., 2007).

Example 10: XCI Status in hESC Lines Derived from Cryopreserved Blastocysts

[00188] In addition to examining XCI in hESC lines that were derived from embryos cultured until the blastocyst stage in physiological oxygen prior to hESC derivation, we also determined the XCI state in several hESC lines derived from embryos cultured to the blastocyst stage under atmospheric oxygen concentrations and then cryopreserved. We thawed 12 cryopreserved blastocysts and allowed the blastocoels to expand (2 blastocysts failed to expand, Table SI) prior to transferring them to physiological oxygen. We obtained three additional hESC lines: 2 female (WIBR4 and WIBR5) and one male (WIBR6) (Figures SI 1 and SI 2). XIST FISH indicated that line WIBR4 was XaXa, while WIBR5 (derived from a blastocyst that failed to expand its blastocoel) had undergone X inactivation (Figure SI 3). This finding demonstrates that it is possible to isolate XaXa hESC lines from embryos frozen at the blastocyst stage, although the additional stress of this procedure (e.g., stress of freeze/thaw at blastocyst stage when inner cell mass has already formed) may result in an increased probability of precocious X chromosome inactivation.

[00189] Experimental Procedures

[00190] Human embryo culture and ICM isolation

[00191] Human embryos at the 8-cell or blastocyst stage produced by in vitro fertilization for clinical purposes were obtained with written informed consent and approved by an MIT institutional review board. Embryos were thawed and cultured in Global medium

(LifeGlobal, Guelph, ON, Canada) supplemented with 15% human Plasmonate (Bayer, Leverkusen, Germany) until day 6 (blastocyst stage) in a low O₂ chamber at 5% O₂, 3% CO₂ and 92% N₂. Embryos were graded as described (Gardner et al., 2000): no blastocoel (grade 2), blastocyst not expanded (grade 3), expanded blastocyst (grade 4), hatching blastocyst (grade 5), hatched blastocyst (grade 6). ICM grade: tightly packed, many cells (grade A), loosely grouped, not many cells (grade B), no ICM visible (grade C). Trophectoderm (TE) was graded as well: many cell forming a cohesive epithelium (grade A), few cells forming a loose epithelium (grade B), very few large cells (grade C).

[00192] ICM isolation was carried out first by removing the zona pelucida (ZP) by 30-40 seconds of treatment with 0.5 mg/ml pronase (Sigma, St. Louis MO) in embryo culture media (LifeGlobal, Guelph, ON, Canada) under microscopic observation. Then, in some

experiments, trophectodermal cells were removed by incubating in human anti-placental alkaline phosphatase antibody diluted 1 : 10 for 40 min. TE cells were lysed by incubating in guinea pig complement (Invitrogen, Carsbad CA), diluted 1 :4 in embryo culture medium and removed using a fire polished Pasteur pipette 100-120 um in diameter. The isolated ICM was then plated in 5% 02 on a feeder layer of freshly plated mitomycin C- inactivated mouse embryonic fibroblasts (MEFs) in human ES cell derivation media. In other experiments, immunosurgery was not used. Instead, whole blastocysts were cultured in human ES cell derivation media (Supplementary Table 1). The hESC derivation media consisted of 75% DMEM/F12 (Invitrogen, Carsbad CA), 7% Fetal Bovine Serum (FBS) (HyClone,

ThermoFischer, Waltham, MA), 7% KO serum replacement (KSR) (Invitrogen, Carsbad CA), 7% human Plasmonate (Bayer, Leverkusen, Germany), 2mM L-glutamine, 1% nonessential amino acids, 50 units/ml penicillin and 50 ug/ml streptomycin (Invitrogen, Carsbad CA), 0.1 mM b-mercaptoethanol (Invitrogen, Carsbad CA), 15 ng/ml ng/ml bFGF (R&D, Minneapolis, MN), 10 μg/ml human LIF (Chemicon, Millipore, Billerica, MA). In some experiments, 20% human serum was used instead of FBS/KSR/Plasmonate (Supplementary Table 1). These procedures were carried out in a 5% O₂ working cabinet (Biospherix, NY, NY). 7-15 days after plating, ICM outgrowth was mechanically dispersed and plated onto two fresh MEF feeder cell layer with hES derivation media (passage 1). One half of the ICM outgrowth was placed in the same physiologic oxygen culture condition (5%O₂, 3%CO₂ and 92%N₂) while the other half was introduced into atmospheric oxygen incubators (20%O₂, 5%CO₂). Secondary colonies resulting from ICM outgrowth were dispersed similarly until passage 2-5. The Plasmonate content of the media and human LIF were eliminated during these initial passages while the content of FBS was increased to 15% and KSR reduced to 5% and bFGF was reduced to 4 ng/ml. This medium was used for continued maintenance of the hESCs. All Whitehead Institute cell lines are named according to their order of derivation as "WIBR1 , 2, 3, 4, 5, 6".

[00193] Culture and in vitro differentiation of hES cells

[00194] All hES lines were maintained on MEFs and passaged mechanically without the use of enzymatic dissociation. For RNA analysis, hES colonies were mechanically isolated and pooled for RNA extraction. Compounds used in oxidative stress protection experiments and cellular stress experiments (Figure 4 and Table S3) were all obtained from Sigma (Sigma, St. Louis MO). For flow cytometric analysis, hES colonies were grown on feeder MEFs constitutively expressing GFP (Hadjantonakis et al., 2001) for a period of 8 days during which colonies became large and exhibited differentiated cells at their periphery. For in vitro differentiation, hESC colonies were harvested with 1 mg/ml collagenase type IV (Invitrogen, Carlsbad, CA), separated from the MEF feeder cells by differential plating, gently triturated, and cultured for 5 days in nonadherent suspension culture dishes (Corning, Lowell, MA) in DMEM supplemented with 20% FBS to promote embryoid body (EB) formation. Small clusters of hESCs were cultured in EB medium (DMEM supplemented with 20% FBS, 2mM L-glutamine, 1% non-essential amino acids, 50 units/ml penicillin and 50 ug/ml streptomycin (Invitrogen, Carsbad CA) on nonadherent culture plates for 5 days to form simple EBs. EBs were then trypsinised to single cells and plated on tissue culture plates and were allowed to differentiate further for 10-12 days.

[00195] Immunocytochemistry of human ES cells

[00196] Cells were fixed in 4% paraformaldehyde in PBS and immunostained according to standard protocols using the following primary antibodies: SSEA4 (mouse monoclonal, Developmental Studies Hybridoma Bank); Tra-1-60, (mouse monoclonal, Chemicon, Millipore, Billerica, MA); SOX2 (goat polyclonal, R&D Systems, Minneapolis, MN); OCT- 3/4 (mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA); hNANOG (goat polyclonal R&D Systems). Appropriate Molecular Probes Alexa Fluor dye-conjugated secondary antibodies (Molecular Probes, Invitrogen, Carlsbad, CA) were used.

[00197] Teratoma formation and analysis

[00198] HES cells were collected by collagenase treatment (1 mg/ml) and separated from feeder cells by subsequent washes with medium and sedimentation of hES cell colonies. HES cell aggregates were collected by centrifugation and resuspended in 100-200 μΐ of hES medium. HES cells were injected subcutaneously into SCID mice (Taconic, Hudson, NY). Tumors generally developed within 6-8 weeks and animals were sacrificed before tumor size exceeded 1.5 cm in diameter. Teratomas were isolated after sacrificing the mice and fixed in formalin, paraffin embedded, and hematoxylin & eosin stained for pathological analysis.

[00199] Karyotype and fingerprint analysis

[00200] Karyotype analysis by G banding was performed by the Cell Line Genetics Laboratory, CLG Inc, Madison, WI, USA.

[00201] Flow cytometry

[00202] For flow cytometric analysis of pluripotency markers, hES colonies were grown on MEFs constitutively expressing the green fluorescent protein. Cells were trypsinized, washed once in PBS and resuspended in FACS buffer (PBS+5% Hyclone FBS). For the SSEA4 staining, 10⁶ cells were stained with l Oul of anti-SSEA4 antibody for 30 minutes, washed lx in FACS buffer, and then stained with an Alexa647 anti-mouse secondary antibody (1 : 100 dilution). One sample was stained with secondary antibody alone. For OCT4 staining, 10⁶ cells were fixed/permeabilized using an intracellular staining kit (R&D systems, Minneapolis, M ). After permeabilization, cells were stained with l Oul of anti- Oct4 primary antibody (Santa Cruz C-10) for 30 minutes, washed once with the kit's wash buffer and stained with an Alexa647 anti-mouse secondary antibody (1 : 100) dilution for 30 minutes. After staining the cells were washed with wash buffer and then resuspended in FACS buffer for analysis on a BD LSR II FACS analyzer. Total human cells were analyzed by gating out GFP-expressing MEF feeders. Data was processed using FlowJo software.

[00203] qRT-PCR and microarray analysis

[00204] For qRT-PCR and Microarray profile analysis, total RNA was isolated from undifferentiated hES colonies between passages 30-40 and isolated using Trizol reagent (Gibco, Invitrogen, Carlsbad, CA) according to the manufacturers protocol. RNA was subsequently treated with DNAse I in order to remove potential genomic DNA contamination using an RNAse free DNAse kit (Zymo Research, Orange County, CA). For qRT-PCR analysis, one mircrogram of DNAse 1 treated RNA was reverse transcribed using a First strand synthesis kit (Invitrogen, Carsbad CA) and ultimately resuspended in lOOul of water. Quantitative PCR analysis was performed in triplicate using 1/50 of the reverse transcription reaction in an ABI Prism 7000 (Applied Biosystems, Foster City, CA) with Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Carsbad CA) using 2-stage cycling parameters. Primers used for XIST amplification were designed spanning introns. Two primer sets were generated spanning 3 different exon-exon boundaries as follows: Exonl -2F: 5'-gaagagtctctggctctttagaatactga-3', Exonl -2R: 5'- cagcgtggtatcttcaatggg-3', Exon5-6F: 5'- gcctggcactctagcacttga-3', Exon5-6R: 5'-aagagacaaagaaatacacattcattcag-3'. To insure equal loading of cDNA into RT reactions, GAPDH mRNA was amplified using F: 5'- TTCACCACCATGGAGAAGGC-3 ' and R: 5'-CCCTTTTGGCTCCACCCT-3\ Data was extracted from in the linear range of amplification and product specificity was verified using melting curve analysis. Analysis of pluripotency and lineage specific genes was carried out using lug of reverse transcribed RNA as described above and amplified using SYBR green and SA Biosciences Human Embryonic Stem Cell PCR arrays and analyzed using RT

Profiler Array Data Analysis software (http://www.sabiosciences.com/pcr/arrayanalysis.php) (SA Biosciences, Frederick MD). Analysis of lineage specific marker gene expression in hES cells both in the undifferentiated state and after 10 days of embryoid body formation was carried out as above using primer sets described in (Soldner et al., 2009).

[00205] For microarray analysis, Cy-dye labeled cRNA samples were prepared using Agilent's QuickAmp sample labeling kit (Agilent, Santa Clara, CA). Input was 2 ug total RNA. Briefly, first and second strand cDNA are generated using MMLV-RT enzyme and an oligo-dT based primer. In vitro transcription is performed using T7 RNA polymerase and either cyanine 3-CTP or cyanine 5-CTP, creating a direct incorporation of dye into the cRNA. Agilent (human 4x44k) expression arrays were hybridized according to our laboratory method, which differs slightly from the Agilent standard hybridization protocol. The hybridization cocktail consisted of 1.65 ug cy-dye labeled cRNA for each sample, Agilent hybridization blocking components, and fragmentation buffer. The hybridization cocktails were fragmented at 60°C for 30 minutes, and then Agilent 2X hybridization buffer was added to the cocktail prior to application to the array. The arrays were then hybridized for 16 hours at 60°C in an Agilent rotor oven set to maximum speed. The arrays were treated with Wash Buffer #1 (6X SSPE / 0.005% n-laurylsarcosine) on a shaking platform at room temperature for 2 minutes, and then Wash Buffer #2 (0.06X SSPE) for 2 minutes at room temperature. The arrays were then dipped briefly in acetonitrile before a final 30 second wash in Agilent Wash 3 Stabilization and Drying Solution, in the hood using a stir plate and stir bar at room temperature. Arrays were scanned using an Agilent scanner and the data was extracted using Agilent's Feature Extraction software set to the default two-color gene expression protocol. [00206] Microarray data processing

[00207] Expression values were processed by within-array LOESS normalization, then by across-array quantile normalization using R package Limma (Smyth and Speed, 2003).

[00208] Gene Expression Plot (Figure 1 A): Different probes representing the same gene were collapsed into a single gene expression value by taking the median among the probe set. Only genes that were expressed (log₂ expression value > 8 in any of the samples) and showed > 2 fold expression change in either 20% Acute or 20% Chronic compared to 5% O₂ were shown. Log₂ expression values relative to 5% reference of each gene are shown as grey lines. Box plot shows the median, lower quartile and upper quartile as well as the 1.5x extreme boundaries of the normalized expression values. Hypoxia-related genes were selected by the gene set HYPOXIA_NORMAL_UP in the Gene Set Enrichment Analysis (GSEA) database (Subramanian et al., 2005).

[00209] Clustering (Figure I B): Hierarchical clustering was performed on the log₂- transformed expression values of all probes that are expressed in at least one sample (log₂ expression value >= 8; 21263 probes) using Cluster3 (de Hoon et al., 2004) (Distance metric: pearson correlation, Linkage method: complete linkage). The tree was visualized in

JavaTreeView (Saldanha, 2004). Significance of clustering was assessed with multiscale bootstrap resampling using R package pvclust (Suzuki and Shimodaira, 2006)

(http://www.is.titech.ac.ip/~shimo/proq/pyclust ). All edges have AU p-value = 100%, demonstrating that the clusters are strongly supported by the data. (Shown on the branches are the pearson correlations).

[00210] Gene Set Enrichment Analysis (Figure S5): Gene Set Enrichment Analysis was performed on the data between 5% and either 20% chronic or 20%» acute with default settings (Subramanian et al., 2005) using the C2 curated all gene matrix

(gseaftp.broad.mit.edu://pub/gsea/gene_sets/c2.all.v2.5. symbols. gmt).

[00211] Heat map of gene expression changes maintained in 20% O₂ (Figure S5): The expression value of each gene was expressed as log₂ expression value relative to 5% sample. The Moderated Welch Test (MWT) (Demissie et al., 2008) was used to find differentially expressed genes between 5% and 20% samples (20%, 20%Acute). Genes with maintained expression from acute to chronic 20% O₂ exposure were selected by requiring FDR<0.1 from the MWT analysis and absolute mean relative log₂ expression value in 20%» acute and 20%» chronic greater than 0.5. A total of 177 genes were selected by these criteria. Average- linkage hierarchical clustering on genes was performed on row (gene)-normalized data using pearson correlation distance metric in cluster3 (de Hoon et al., 2004). Heatmap visualization was generated using JavaTreeView (Saldanha, 2004).

[00212] RNA/DNA FISH and immunofluorescence

[00213] RNA and DNA FISH was carried out as described in (Lee and Lu, 1999).

Dispersed hESCs were cytospun onto glass slides prior to fixation. cDNA probes were generated to AZST exon 1 (GenBank U80460: 61251-69449)and exon 6 (U80460: 75081- 78658), labeled by nick translation (Roche, Indianapolis, IN) with Cy3-dUTP (Amersham) and Cot-1 DNA was labeled with fluoroscein-12-dUTP using the Prime-It Fluor Labeling kit (Stratagene, La Jolla, CA). After RNA FISH was performed, 0.2-μπι z-section images were captured and the z-sections were merged for images StarFISH X chromosomal paints (Cambio, Cambridge, UK) were hybridized according to manufacturer's instructions. In sequential RNA/DNA FISH, RNA FISH was performed first, 0.2-μηι z-section images were captured, their x-y coordinates were marked, and then the same slide was denatured for subsequent DNA FISH. For X-linked gene FISH, a BAC containing the VBPl genomic locus was labeled by nick translation (Roche, Indianapolis, IN) with Cy3-dUTP (Amersham) and hybridization/imaging was performed as for XIST. Images were then overlaid using DAPI nuclear staining as a reference.

[00214] Imprinted and XIST methylation analysis

[00215] Conversion of genomic DNA with sodium bisulfite was performed using EZ-96 DNA Methylation Kit (Zymo Research, Orange County, CA) with 1 ug of genomic DNA and the alternative conversion protocol (a two temperature DNA denaturation). Sequenom's MassARRAY platform was used to perform quantitative methylation analysis. This system utilizes matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern is then analyzed for methylation status. PCR primers were derived from the sequenom imprinting Epipanel (http://www.sequenom.com/getdoc/6e97d7f2-3499-433d- a44d-fb73685af93a/Imprinting-EpiPanel). For XIST methylation analysis, a total of four ampl icons across a lkb region upstream of the transcriptional start site were generated using the following primer sequences: amplicon 1 L 5'-GGA TAT TTG TTT TAA TTT TTG TTT TTT TGG-3', amplicon 1R 5'-AAC TTA ACT ACA AAA TCA TTC TCT ACC A-3', amplicon 7L 5'-TGT TTT AGA AAG AAT TTT AAG TGT AGA GA-3', amplicon 7R 5'- AAA TAA ATT TTA AAC CAA CCA AAT CAC-3', amplicon 9F 5'-TAG AGG GGA AGG GAA TTA GTA GG TAT T-3' amplicon 9R 5'- AAA CTA AAA ACT TCC TAA CTA AAA ATC TC-3', amplicon 1 IF 5'-TGG GTT AGA AAA ATA AAA ATT AAA GTA GG-3', amplicon 1 1R 5'-AAT ACC TAC TAA TTC CCT TCC CCT CTA-3'. Reverse primers contain a T7 promoter binding site for in vitro transcription. The MassCLEAVE biochemistry was performed as previously described (Ehrich et al., 2005). Mass spectra were acquired using a MassARRAY Compact MALDI-TOF (Sequenom, San Diego, CA) and spectra's methylation ratios were generated by the Epityper software vl .O (Sequenom).

[00216] Allele-specific expression analysis: SNP Chips.

[00217] The allele-specific analysis of the whole X chromosomes was performed on the RNA from cell nuclei as described in (Gimelbrant et al., 2007). Nuclei from the cells were prepared using NucleiPure kit (Sigma, St. Louis MO) by gentle cell lysis followed by sucrose cushion centrifugation. The preparations started with 3-10 million cells. Nuclei were stored in NucleiPure storage solution (Sigma, St. Louis MO) at -80°C. Total RNA was extracted from the nuclei using Trizol reagent (Roche, Indianapolis, IN), then diluted appropriately and treated with Turbo DNAfree (Ambion, Foster City, CA) according to manufacturer's protocol for "strong DNA contamination". Genomic DNA from the nuclei was prepared with

QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA).

[00218] The DNA-free total RNA from nuclei was converted to double stranded cDNA as described in (Holstege et al., 1998). Briefly, reverse transcription of 3 ug of RNA (37°C, Superscript II; Invitrogen, Carsbad CA) was followed by second strand synthesis using a mix of DNA polymerase I, DNA ligase, and RNAse H (Invitrogen, Carsbad CA). The resulting DNA was cleaned up by phenol-chloroform extraction and ethanol precipitation and resuspended in low-EDTA TE buffer (TekNova, Hollister, CA). Genomic DNA was resuspended at 50 ng/ul, and cDNA at 100 ng/ul, quantified by optical absorption.

[00219] The DNA and cDNA samples were further processed according to the standard protocol for the 250K Nsp Human Mapping Array (Affymetrix, Santa Clara, CA). After hybridization, genotypes were called by the Affymetrix GTYPE software using Dynamic Model Mapping algorithm (DM). For genomic DNA samples, all genotypes, as called by DM, were accepted. For cDNA samples, we accepted only the genotypes that were replicated with DM confidence score less than 0.15. The downstream data summarization and visualization were performed as described in (Gimelbrant et al., 2007). [00220] Allele-specific expression analysis: Sequenom genotyping

[00221] Sequenom genotyping can be used for precise measurement of allelic imbalance in DNA or cDNA samples {see (Cowles et al., 2002; Gimelbrant and Chess, 2006), and references therein} . This methodology involves PCR amplification of a small region flanking the SNP of interest, followed by primer extension and mass spectrometric detection of extended species.

[00222] The singleplex genotyping assays were designed using Sequenom online design pipeline with process, with 5-10 ng of genomic DNA or double-stranded cDNA (default parameters, starting with dbSNP RS numbers for SNPs of interest. We used hME prepared as described above) as the initial PCR template. Quantitative allele frequency mode was used for the mass spectra collection. Each assay was run in quadruplicate, and the average of the four assays was used for further calculations.

[00223] Chromatin Immunoprecipitation and Sequencing (ChlP-Seq)

[00224] The antibodies for ChIP were specific for H3K4me3 (ab 8580) and H3K27me3 (ab 6002). Protocols describing all materials and methods have been previously described and can be downloaded from http://web.wi.mit.edu/young/hES_PRC and are described in detail (Lee et al., 2006).

7 8

[00225] Human stem cell lines were grown to a final count of 5x10 - 1x10 cells for each chromatin immunoprecipitation (ChIP). Cells were chemically crosslinked by the addition of one-tenth volume of fresh 1 1% formaldehyde solution for 15 minutes at room temperature. Cells were rinsed twice with lxPBS, harvested by centrifugation and flash frozen in liquid nitrogen. Cells were stored at -80°C prior to use. Cells were resuspended, lysed and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking and equipment. We used a Misonix Sonicator 3000 and sonicated at a power of 27W for 10 x 30 second pulses (90 second pause between pulses). Samples were kept on ice at all times.

[00226] The resulting whole cell extract (1/6 of total lysate which equals approximately 1 million cells) was incubated overnight at 4°C with 10 μΐ of Dynal Protein G magnetic beads that had been preincubated with 5 μg of the appropriate antibody. The immunoprecipitation was allowed to proceed overnight. Beads were washed 5 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Immunoprecipitated DNA was then purified by treatment with RNAse A, proteinase and multiple phenol:chloroform:isoamyl alcohol extractions.

[00227] All protocols for Illumina/Solexa sequence preparation, sequencing and quality control are provided by Illumina (Illumina, San Diego, CA,

http://www.illumina.com/pages. ilmn?ID=252). A brief summary of the technique as well as minor protocol modifications in addition to data analysis methods are described below.

[00228] Sample Preparation

[00229] Purified chromatin immunoprecipitated (ChIP) DNA was prepared for sequencing according to a modified version of the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was prepared for ligation of Solexa linkers by repairing the ends and adding a single adenine nucleotide overhang to allow for directional ligation. A 1 : 100 dilution of the Adaptor Oligo Mix (Illumina) was used in the ligation step. A subsequent PCR step with limited (18) amplification cycles added additional linker sequence to the fragments to prepare them for annealing to the Genome Analyzer flow-cell. After amplification, a narrow range of fragment sizes was selected by separation on a 2% agarose gel and excision of a band between 150-300 bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence). The DNA was purified from the agarose and diluted to 10 nM for loading on the flow cell.

[00230] Polony Generation on Solexa Flow-Cells and Sequencing

[00231] The DNA library (2-4 pM) was applied to the flow-cell (eight samples per flow- cell) using a Cluster Station device (Illumina). The concentration of library applied to the flow-cell was calibrated so that polonies generated in the bridge amplification step originate from single strands of DNA. Multiple rounds of amplification reagents were flowed across the cell in the bridge amplification step to generate polonies of approximately 1 ,000 strands in 1 μm diameter spots. Double stranded polonies were visually checked for density and morphology by staining with a 1 :5000 dilution of SYBR Green I (Invitrogen) and visualizing with a microscope under fluorescent illumination. Validated flow-cells were stored at 4°C until sequencing. Flow-cells were removed from storage and subjected to linearization and annealing of sequencing primer on the Cluster Station. Primed flow-cells were loaded into the Genome Analyzer 1 G (Illumina). After the first base was incorporated in the sequencing- by-synthesis reaction the process was paused for a key quality control checkpoint. A small section of each lane was imaged and the average intensity value for all four bases was compared to minimum thresholds. Flow-cells with low first base intensities were re-primed and if signal was not recovered the flow-cell was aborted. Flow-cells with signal intensities meeting the minimum thresholds were resumed and sequenced for 26 cycles.

[00232] Genomic Mapping of ChlP-Seq Reads

[00233] Images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline that identified polony positions, performed base- calling and generated QC statistics. Sequences were aligned using ELAND software to NCBI Build 36 (UCSC mm8) of the mouse genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple reads mapped to the same genomic position, a maximum of two reads mapping to the same position were used. Sequence reads from multiple flow cells for each IP target were combined. Each read was extended lOObp, towards the interior of the sequenced fragment, based on the strand of the alignment. Across the genome, in 25 bp bins, the number of ChlP-Seq reads within a lkb window surrounding each bin (+/- 500bp) was tabulated. Enriched bins within l kb of one another were combined into regions. The complete set of RefSeq genes was downloaded from the UCSC table browser (http://genome.ucsc.edu/cgi-bin/hgTables?command=^:start) on December 20, 2008. Genes with enriched regions within 4 kb of their transcription start site were called bound.

[00234] Normalization

[00235] In order to facilitate comparison ChlP-Seq datasets across the three cell lines a quantile normalization method was used. Across all datasets the genomic bin with the greatest ChlP-Seq density was identified. The average of these values was calculated and the bin with the highest signal in each dataset was assigned this average value. This was repeated for all genomic bins from the greatest signal to the least, assigning each the average ChlP-Seq signal for all bins of that rank across all datasets.

[00236] ChlP-Seq Density Heatmaps

[00237] Genes were aligned with each other according to the position and direction of their transcription start site. For each experiment, the ChlP-Seq density profiles were normalized to the density per million total reads. Genes were sorted as indicated. Heatmaps were generated using Java Treeview (http://jtreeview.sourceforge.net/) with color saturation as indicated in the figures and legends.

* * * [00238] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the embodiments described above. The invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[00239] Articles such as "a" and "an", and the like, may mean one or more than one unless indicated to the contrary or otherwise evident from the context.

[00240] The phrase "and/or" as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when used in a list of elements, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but optionally more than one, of list of elements, and, optionally, additional unlisted elements. Only terms clearly indicative to the contrary, such as "only one of "or "exactly one of will refer to the inclusion of exactly one element of a number or list of elements. Thus claims that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process unless indicated to the contrary. The invention provides embodiments in which exactly one member of the group is present, employed in, or otherwise relevant to a given product or process. The invention also provides embodiments in which more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process. It is to be understood that the invention encompasses embodiments in which one or more limitations, elements, clauses, descriptive terms, etc., of a claim is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more elements or limitations found in any other claim that is dependent on the same base claim. [00241] Where the claims recite a composition, it is understood that methods of using the composition as disclosed herein are provided, and methods of making the composition according to any of the methods of making disclosed herein are provided. Where the claims recite a method, it is understood that a composition for performing the method is provided. Where elements are presented as lists or groups, each subgroup is also disclosed. It should also be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc.

[00242] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[00243] Where ranges are given herein, the invention provides embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and

embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. "About" in reference to a numerical value generally refers to a range of values that fall within ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5% of the value unless otherwise stated or otherwise evident from the context. In any embodiment of the invention in which a numerical value is prefaced by "about", the invention provides an embodiment in which the exact value is recited. In any embodiment of the invention in which a numerical value is not prefaced by "about", the invention provides an embodiment in which the value is prefaced by "about". Where the phrase "at least" precedes a series of numbers, it is to be understood that the phrase applies to each number in the list (it being understood that, depending on the context, 100% of a value may be an upper limit). It is also understood that any particular embodiment, feature, or aspect of the present invention may be explicitly excluded from any one or more of the claims. For example, any one or more antioxidants, test compounds, or mammalian species may be excluded.

REFERENCES

Batt, P.A., Gardner, D.K., and Cameron, A.W. (1991 ). Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod Fertil Dev 3, 601 -607.

Bernardi, M.L., Flechon, J.E., and Delouis, C. (1996). Influence of culture system and oxygen tension on the development of ovine zygotes matured and fertilized in vitro. J Reprod Fertil 106, 161 -167.

Boggs, B.A., Cheung, P., Heard, E. , Spector, D.L., Chinault, A.C., and Allis, CD. (2002). Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes. Nat Genet 30, 73-76.

Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B. , Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L, Pedersen, R.A., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191 -195.

Chen, H.F., Kuo, H.C., Chen, W., Wu, F.C., Yang, Y.S., and Ho, H.N. (2009). A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals. Hum Reprod 24, 71 -80.

Cowan, C.A., Klimanskaya, I., McMahon, J., Atienza, J., Witmyer, J., Zucker, J. P., Wang, S., Morton, C.C., McMahon, A.P., Powers, D., er al. (2004). Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 350, 1353-1356.

Cowles, C.R., Hirschhorn, J.N., Altshuler, D., and Lander, E.S. (2002). Detection of regulatory variation in mouse genes. Nat Genet 32, 432-437.

de Hoon, M.J., Imoto, S., Nolan, J., and Miyano, S. (2004). Open source clustering software. Bioinformatics 20, 1453-1454.

Demissie, M., Mascialino, B., Calza, S., and Pawitan, Y. (2008). Unequal group variances in microarray data analyses. Bioinformatics 24, 1 168-1 174.

Ehrich, M., Nelson, M.R., Stanssens, P., Zabeau, M., Liloglou, T., Xinarianos, G., Cantor, C.R., Field, J.K., and van den Boom, D. (2005). Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci U S A 102, 15785-15790.

Ezashi, T., Das, P., and Roberts, R.M. (2005). Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A 102, 4783-4788.

Fischer, B., and Bavister, B.D. (1993). Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil 99, 673-679. Forsyth, N.R., Kay, A., Hampson, K., Downing, A., Talbot, R., and McWhir, J. (2008). Transcriptome alterations due to physiological normoxic (2% 02) culture of human embryonic stem cells. Regen Med 3, 817-833.

Forsyth, N.R., Musio, A., Vezzoni, P., Simpson, A.H., Noble, B.S., and McWhir, J. (2006). Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning Stem Cells 8, 16-23.

Gardner, D.K., Lane, M., Stevens, J., Schlenker, T., and Schoolcraft, W.B. (2000). Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 73, 1 155-1 158.

Gimelbrant, A., Hutchinson, J.N. , Thompson, B.R. , and Chess, A. (2007). Widespread monoallelic expression on human autosomes. Science 318, 1 136-1 140.

Gimelbrant, A.A., and Chess, A. (2006). An epigenetic state associated with areas of gene duplication. Genome Res 16, 723-729.

Guo, G., Yang, J., Nichols, J. , Hall, J.S., Eyres, I., Mansfield, W., and Smith, A. (2009). Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063-1069.

Hadjantonakis, A.K., Cox, L.L., Tarn, P.P., and Nagy, A. (2001 ). An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta. Genesis 29, 133-140.

Hall, L.L., Byron, M., Butler, J., Becker, K.A., Nelson, A., Amit, M., Itskovitz-Eldor, J., Stein, J., Stein, G. , Ware, C, et al. (2008). X-inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected. J Cell Physiol 216, 445-452.

Harlow, G.M., and Quinn, P. (1979). Foetal and placental growth in the mouse after pre-implantation development in vitro under oxygen concentrations of 5 and 20%. Aust J Biol Sci 32, 363-369.

Heard, E., Rougeulle, C, Arnaud, D., Avner, P., Allis, CD., and Spector, D.L. (2001 ). Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell 107, 727-738.

Holstege, F.C., Jennings, E.G., Wyrick, J.J. , Lee, T.I., Hengartner, C.J., Green, M.R., Golub, T.R., Lander, E.S. , and Young, R.A. (1998). Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717-728.

Huynh, K.D., and Lee, J.T. (2003). Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature 426, 857-862.

Kalantry, S., Purushothaman, S., Bowen, R.B., Starmer, J., and Magnuson, T. (2009). Evidence of Xist RNA-independent initiation of mouse imprinted X- chromosome inactivation. Nature 460, 647-651. Karja, N.W., Wongsrikeao, P., Murakami, M., Agung, B., Fahrudin, M., Nagai, T., and Otoi, T. (2004). Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos. Theriogenology 62, 1585-1595.

Kay, G.F., Penny, G.D., Patel, D., Ashworth, A., Brockdorff, N., and Rastan, S. (1993). Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 72, 171 -182.

Kikuchi, K., Onishi, A., Kashiwazaki, N., Iwamoto, M., Noguchi, J., Kaneko, H., Akita, T., and Nagai, T. (2002). Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol Reprod 66, 1033-1041.

Kitagawa, Y., Suzuki, K., Yoneda, A., and Watanabe, T. (2004). Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology 62, 1 186-1 197.

Kohlmaier, A. , Savarese, F., Lachner, M., Martens, J., Jenuwein, T. , and Wutz, A. (2004). A chromosomal memory triggered by Xist regulates histone methylation in X inactivation. PLoS Biol 2, E171.

Lee, J.T., and Lu, N. (1999). Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99, 47-57.

Lee, T.I., Johnstone, S.E., and Young, R.A. (2006). Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1, 729-748.

Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T., Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R., Piekarczyk, M.S., ef al. (2006). Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24, 185-187.

Mak, W., Nesterova, T.B., de Napoles, M., Appanah, R., Yamanaka, S., Otte, A.P., and Brockdorff, N. (2004). Reactivation of the paternal X chromosome in early mouse embryos. Science 303, 666-669.

Mermoud, J.E., Popova, B., Peters, A.H., Jenuwein, T., and Brockdorff, N. (2002). Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation. Curr Biol 12, 247-251.

Monkhorst, K., Jonkers, I., Rentmeester, E., Grosveld, F., and Gribnau, J. (2008). X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator. Cell 132, 410-421.

Nesterova, T.B., Popova, B.C., Cobb, B.S., Norton, S., Senner, C.E., Tang, Y.A., Spruce, T., Rodriguez, T.A., Sado, T., Merkenschlager, M., et al. (2008). Dicer regulates Xist promoter methylation in ES cells indirectly through transcriptional control of Dnmt3a. Epigenetics Chromatin 1, 2. Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487-492.

Okamoto, I., Otte, A.P., Allis, CD., Reinberg, D., and Heard, E. (2004). Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303, 644-649.

Panning, B., Dausman, J., and Jaenisch, R. (1997). X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907-916.

Parrinello, S., Samper, E., Krtolica, A., Goldstein, J., Melov, S., and Campisi, J. (2003). Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5, 741-747.

Patrat, C, Okamoto, I., Diabangouaya, P., Vialon, V., Le Baccon P., Chow, J., and Heard, E. (2009). Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc Natl Acad Sci U S A 106, 5198- 5203.

Payer, B., and Lee, J.T. (2008). X chromosome dosage compensation: how mammals keep the balance. Annu Rev Genet 42, 733-772.

Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S., and Brockdorff, N. (1996). Requirement for Xist in X chromosome inactivation. Nature 379, 131 -137.

Plath, K., Fang, J., Mlynarczyk-Evans, S.K. , Cao, R., Worringer, K.A., Wang, H. , de la Cruz, C.C., Otte, A.P., Panning, B., and Zhang, Y. (2003). Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131-135.

Prasad, S.M., Czepiel, M., Cetinkaya, C, Smigielska, K., Weli, S.C., Lysdahl, H., Gabrielsen, A., Petersen, K., Ehlers, N., Fink, T., et al. (2009). Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif 42, 63-74.

Quinn, P., and Harlow, G.M. (1978). The effect of oxygen on the development of preimplantation mouse embryos in vitro. J Exp Zool 206, 73-80.

Reubinoff, B.E., Pera, M.F., Fong, C.Y. , Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399-404.

Saldanha, A.J. (2004). Java Treeview-extensible visualization of microarray data. Bioinformatics 20, 3246-3248.

Shen, Y., Matsuno, Y., Fouse, S.D., Rao, N., Root, S., Xu, R., Pellegrini, M., Riggs, A.D., and Fan, G. (2008). X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. Proc Natl Acad Sci U S A 105, 4709-4714. Silva, S.S., Rowntree, R.K., Mekhoubad, S., and Lee, J.T. (2008). X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proc Natl Acad Sci U S A 105, 4820-4825.

Smyth, G.K., and Speed, T. (2003). Normalization of cDNA microarray data. Methods 31, 265-273.

Soldner, F., Hockemeyer, D., Beard, C, Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., et al. (2009). Parkinson's disease patient- derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977.

Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545-15550.

Sun, B.K., Deaton, A.M., and Lee, J.T. (2006). A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol Cell 21, 617-628.

Suzuki, R., and Shimodaira, H. (2006). Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 1540-1542.

Tesar, P. J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196-199.

Thompson, J.G., Simpson, A.C., Pugh, P.A., Donnelly, P.E., and Tervit, H.R. (1990). Effect of oxygen concentration on in-vitro development of preimplantation sheep and cattle embryos. J Reprod Fertil 89, 573-578.

Thomson, J.A., Itskovitz-Eldor, J. , Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1 145-1 147.

van den Berg, I.M., Laven, J.S., Stevens, M., Jonkers, I., Galjaard, R.J., Gribnau, J., and van Doorninck, J.H. (2009). X chromosome inactivation is initiated in human preimplantation embryos. Am J Hum Genet 84, 771-779.

Wang, F., Thirumangalathu, S., and Loeken, M.R. (2006). Establishment of new mouse embryonic stem cell lines is improved by physiological glucose and oxygen. Cloning Stem Cells 8, 108-1 16.

Ware, C.B., Wang, L., Mecham, B.H. , Shen, L., Nelson, A.M., Bar, M., Lamba, D.A., Dauphin, D.S., Buckingham, B., Askari, B., et al. (2009). Histone deacetylase inhibition elicits an evolutionary conserved self-renewal program in embryonic stem cells. Cell Stem Cell 4, 359-369. Wutz, A. , and Jaenisch, R. (2000). A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol Cell 5, 695-705.

Wutz, A., Rasmussen, TP., and Jaenisch, R. (2002). Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat Genet 30, 167-174.

Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., and Yamanaka, S. (2009). Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5, 237-241 .

Zhang, L.F., Huynh, K.D., and Lee, J.T. (2007). Perinucleolar targeting of the inactive X during S phase: evidence for a role in the maintenance of silencing. Cell 129, 693-706.

Claims

We claim:

1. A pluripotent human embryonic stem (ES) cell line derived under physiological oxygen conditions.

2. The pluripotent human ES cell line of claim 1, wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%.

3. The pluripotent human ES cell line of claim 1 , wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%, a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%.

4. The pluripotent human ES cell line of claim 1, wherein the cell line has a normal karyotype.

5. The pluripotent human ES cell line of claim 1, wherein the cell line proliferates in culture and maintains pluripotency for at least one year.

6. The pluripotent human ES cell line of claim 1 , wherein the cell line proliferates in culture in a genetically stable manner for at least one year.

7. The pluripotent human ES cell line of claim 1, wherein the cell line (a) contains two X chromosomes; (b) is negative for XIST; and (c) has the ability to activate XIST expression upon differentiation.

8. The pluripotent human ES cell line of claim 7, wherein the X chromosomes are active.

9. The pluripotent human ES cell line of claim 7, wherein the cell line exhibits biallelic expression of at least 90% of informative X-linked SNPs.

10. The pluripotent human ES cell line of claim 7, wherein one of said two X

chromosomes becomes randomly inactivated upon differentiation.

1 1. The pluripotent human ES cell line of claim 7, wherein the cell line proliferates in culture in a genetically stable manner, remains negative for XIST, and retains the ability to activate XIST expression upon differentiation, for at least one year.

12. A cell, colony, or subclone of the pluripotent human ES cell line of claim 7.

13. A cell, colony, or subclone of the pluripotent human ES cell line of claim 1.

14. A non-pluripotent cell descended from a cell of the human ES cell line of claim 1.

15. A non-pluripotent descendant of a cell of the human ES cell line of claim 1 , wherein the non-pluripotent descendant is obtained under physiological 02 conditions.

16. A cell culture comprising non-pluripotent descendants of the cell line of claim 1, wherein cells of the cell culture contain 2 X chromosomes, and wherein the cell culture exhibits biallelic expression of X-linked genes.

17. A composition comprising isolated pluripotent human ES cells that were derived under physiological oxygen conditions.

18. The composition of claim 17, wherein the pluripotent human ES cells (a) contain two X chromosomes; (b) are negative for XIST; and (c) have the ability to activate XIST expression upon differentiation.

19. The composition of claim 17, wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%.

20. The composition of claim 17, wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%, a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%.

21. The composition of claim 17, further comprising a culture medium suitable for culturing human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

22. The composition of claim 17, further comprising a culture medium suitable for culturing human ES cells, wherein the medium comprises at least one antioxidant.

23. The composition of claim 17, further comprising a culture medium suitable for culturing human ES cells, wherein the medium comprises at least one antioxidant selected from the group consisting of: vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

24. A method of deriving a cell line comprising steps of:

(a) obtaining an isolated early stage human embryo; and

(b) deriving a pluripotent human ES cell line from the isolated early stage human embryo under physiological oxygen conditions.

25. The method of claim 24, wherein the isolated early stage human embryo of step (a) is a blastocyst.

26. The method of claim 24, wherein the method comprises steps of:

i. obtaining an isolated human embryo at or before the 8 cell stage;

ii. allowing the isolated human embryo to develop to a blastocyst in culture under physiological oxygen conditions; and

iii. deriving a pluripotent human ES cell from the blastocyst under physiological oxygen conditions.

27. The method of claim 25 or 26, wherein the method comprises:

(a) isolating cells from the inner cell mass (ICM) of the blastocyst;

(b) plating the ICM cells under suitable conditions such that ICM-derived outgrowths develop;

(c) dispersing the ICM-derived outgrowths to obtain dispersed cells;

(d) replating the dispersed cells under conditions suitable for development of hES cell colonies; and

(e) culturing cells of one or more of the colonies to obtain a pluripotent human ES cell line, wherein steps (a)-(e) are performed under physiological oxygen conditions.

28. The method of claim 27, wherein suitable conditions of step (b) comprise culturing the cells on an embryonic feeder cell layer.

29. The method of claim 27, wherein the cells are passaged mechanically without use of enzymatic dissociation.

30. The method of claim 24, wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%.

31. The method of claim 24, wherein the physiological oxygen conditions comprise an oxygen concentration of about 5%, a carbon dioxide concentration of about 3%, and a nitrogen concentration of about 92%.

32. The method of claim 24, wherein the early stage embryo has two X chromosomes.

33. The method of claim 24, further comprising culturing the derived ES cell line in medium suitable for culturing human ES cells for at least two passages, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

34. A method of deriving a cell line comprising steps of:

(a) obtaining an isolated early stage human embryo; and

(b) deriving a pluripotent human ES cell line from the isolated early stage human embryo under conditions that mimic the effect of physiological 02 conditions.

35. The method of claim 34, wherein the conditions that mimic the effect of

physiological 02 conditions are sufficient to preserve the XaXa status of XaXa ESCs derived under physiological 02 conditions following shift to atmospheric 02 conditions.

36. The method of claim 34, wherein the isolated early stage human embryo of step (a) is a blastocyst.

37. The method of claim 34, wherein the method comprises steps of:

i. obtaining an isolated human embryo at or before the 8 cell stage;

ii. allowing the isolated human embryo to develop to a blastocyst in culture under conditions that mimic the effect of physiological 02 conditions; and

iii. deriving a pluripotent human ES cell from the blastocyst under conditions that mimic the effect of physiological 02 conditions.

38. The method of claim 35 or 37, wherein the method comprises:

(a) isolating cells from the inner cell mass (ICM) of the blastocyst; (b) plating the ICM cells under suitable conditions such that ICM-derived outgrowths develop;

(c) dispersing the ICM-derived outgrowths to obtain dispersed cells;

(e) culturing cells of one or more of the colonies to obtain a pluripotent human ES cell line, wherein steps (a)-(e) are performed under conditions that mimic physiological 02 conditions.

39. The method of claim 38, wherein suitable conditions of step (b) comprise culturing the cells on an embryonic feeder cell layer.

40. The method of claim 38, wherein the cells are passaged mechanically without use of enzymatic dissociation.

41. The method of claim 34, wherein the conditions that mimic physiological 02 conditions comprise culture in medium comprising at least one compound that mimics the effect of physiological 02 conditions.

42. The method of claim 34, wherein the conditions that mimic physiological 02 conditions comprise culture in medium comprising at least one antioxidant.

43. The method of claim 34, wherein the conditions that mimic physiological 02 conditions comprise culture in medium comprising at least one antioxidant selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

44. The method of claim 34, wherein the early stage embryo has two X chromosomes.

45. The method of claim 34, further comprising culturing the derived cell line in medium suitable for culturing a human ES cell, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

46. A method of producing a population of human ES cells comprising steps of: (a) providing pluripotent human ES cells derived under physiological oxygen conditions; and

(b) culturing the pluripotent human ES cells in medium suitable for culturing pluripotent human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

47. The method of claim 46, wherein the culture medium comprises at least one antioxidant.

48. The method of claim 46, wherein the culture medium comprises at least one antioxidant selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

49. The method of claim 46, wherein the pluripotent human ES cells of step (b) are cultured under standard oxygen conditions.

50. The method of claim 46, wherein the pluripotent human ES cells of step (a) have two X chromosomes and are negative for XIST expression, and wherein the population of step (b) remains negative for XIST expression for at least 10 passages when cultured in said medium.

51. The method of claim 46, wherein the pluripotent human ES cells of step (a) have two active X chromosomes, and wherein the population of step (b) retains two active X chromosomes for at least 10 passages when cultured in said medium.

52. A cell culture medium suitable for deriving or culturing human ES cells, wherein the medium comprises at least one compound that mimics the effect of physiological 02 conditions.

53. The cell culture medium of claim 52, wherein the compound is present in an amount sufficient to preserve the XaXa status of hESC derived under physiological 02 conditions following shift to atmospheric 02 conditions.

54. The cell culture medium of claim 52, wherein the compound comprises at least one antioxidant.

55. The cell culture medium of claim 52, wherein the medium comprises at least one antioxidant selected from the group consisting of vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

56. A kit comprising: ingredients for a cell culture medium suitable for deriving or culturing human pluriopotent cells, wherein the ingredients include at least one compound in an amount sufficient to mimic the effect of physiological 02 conditions, and wherein the kit optionally comprises at least one item selected from the group consisting of: (i) instructions for preparing the medium; (ii) instructions for deriving or culturing human pluripotent cells; (iii) serum replacement; (iv) albumin; (v) at least one protein or small molecule useful for deriving or culturing human ES cells, wherein the protein or small molecule activates or inhibits a signal transduction pathway; (vi) at least one reagent useful for characterizing human pluripotent cells; and (vii) pluripotent human cells, which are optionally ESCs of the invention..

57. The kit of claim 56 wherein the at least one compound that mimics the effect of

physiological 02 conditions is an antioxidant.

58. The kit of claim 56 wherein the at least one compound that mimics the effect of physiological 02 conditions is an antioxidant selected from the group consisting of: vitamin C, vitamin E, lipoic acid, morin hydrate, and combinations thereof.

59. The kit of claim 56 wherein at least some of the ingredients are dissolved in liquid.

60. The kit of claim 56 wherein at least some of the ingredients are provided in dry form.