WO2008134522A1 - Dérivation de cellules souches embryonnaires - Google Patents

Dérivation de cellules souches embryonnaires Download PDF

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WO2008134522A1
WO2008134522A1 PCT/US2008/061591 US2008061591W WO2008134522A1 WO 2008134522 A1 WO2008134522 A1 WO 2008134522A1 US 2008061591 W US2008061591 W US 2008061591W WO 2008134522 A1 WO2008134522 A1 WO 2008134522A1
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cell
cells
nuclear
transgenic
donor
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Kevin Eggan
Dieter Egli
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8775—Murine embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0273—Cloned vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/04—Cells produced using nuclear transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • the present invention provides methods useful for the generation a transgenic cell.
  • methods disclosed herein comprise removing nuclear-derived genetic material from a fertilized zygote or a blastomere and introducing nuclear-derived genetic material derived from a donor cells.
  • such a fertilized zygote or a blastomere is arrested in mitosis such that the nuclear envelope has broken down prior to removing the nuclear-derived genetic material.
  • nuclear-derived genetic material derived from a donor cell is introduced into the fertilized zygote or a blastomere via injection. In certain embodiments, nuclear-derived genetic material derived from a donor cell is introduced into the fertilized zygote or a blastomere via fusion of the donor cell with the fertilized zygote or a blastomere.
  • the present invention encompasses the recognition of the possibility that there are factors required for reprogramming and/or embryonic development (Do, J. T. & Scholer, H. R., Nuclei of embryonic stem cells reprogram somatic cells, Stem Cells 22, 941-9, 2004; Takahashi, K. & Yamanaka, S.,
  • mouse zygotes were reversibly arrested in mitosis, their chromosomes removed and replaced with chromosomes from either embryonic or somatic donor cells. It was discovered that such reconstructed zygotes can develop into blastocysts and that these blastocysts can be used to generate cloned animals and ES cell lines.
  • Figure 1 shows the first embryonic cell cycle
  • a Diagram of the first cell cycle.
  • condensed chromosomes are aligned on a metaphase plate, while nuclear factors that might be required for reprogramming or development are dispersed throughout the cytoplasm.
  • the parental genomes and required nuclear factors are sequestered in the pronuclei.
  • the factors should be released into the cytoplasm, b, oocyte arrested in metaphase of meiosis II.
  • c Zygote in interphase with two pronuclei, d, zygote arrested in prometaphase of mitosis by nocodazole.
  • FIG. 2 shows one embodiment of CT into zygotes arrested in mitosis, a, Diagram outlining the method of chromosome transfer into zygotes arrested in mitosis. b, Arrested zygotes before spindle and chromosome removal, 10 min after the shift from nocodazole to MG-132.
  • d Removal of the zygote spindle and chromosomes by micromanipulation, d, A large group of zygotes after spindle removal all lack chromosomes, e, Piezo actuated injection of a nocodazole arrested embryonic stem (ES) cell, into a mitotic zygote .
  • ES nocodazole arrested embryonic stem
  • f ES cell chromosomes in the zygote immediately after the transfer, g, 80 min after transfer a new spindle has formed and the chromosomes have aligned in a new metaphase plate, h, i, Progression through the first mitosis following ES cell chromosome transfer, h, From left to right: prometaphase/metaphase, anaphase, and telophase/cytokinesis. i, Equal chromosome segregation into the two daughter blastomeres.
  • Figure 3 shows developmental potential in vitro and in vivo after ES cell CT into mitotic zygotes, a, Experimental outline, b, H2B-cherry donor ES cells with and without induction of the transgene by doxycycline.
  • ICM inner cell mass
  • h Primary culture of skin from a cloned pup.
  • neo neomycin
  • j Bar diagram showing weight of pups and their placentas produced by CT with ES cells, mitotic spindles of either another zygote or of a 2-cell stage blastomere, and of controls
  • Crossbars indicate standard deviations.
  • Figure 4 shows one embodiment of derivation of ES cell lines from somatic cell chromosome transfer blastocysts, a, Diagram of zygote somatic cell chromosome transfer (SCCT) and derivation of SCCT ES cells, b, Somatic donor cell line with fibroblast morphology, c, Zygote SCCT blastocyst, d, Zygote SCCT ICM outgrowth 16 days after plating, e, Zygote SCCT ES cell culture, f, g, Expression of the pluripotency marker gene Oct4 and the embryonic antigen SSEA-I in zygote SCCT ES cells, detected by immunostaining.
  • SCCT somatic cell chromosome transfer
  • Figure 5 shows that aneuploid zygotes with more than two pronuclei can be used as recipients for chromosome transfer
  • a Diagram of an IVF reaction without a zona pellucida. The increased access to sperm results in a high frequency of polyspermic zygotes
  • b Dispermic zygote in interphase with two paternal pronuclei and a single maternal pronucleus (arrows)
  • c Dispermic zygote after pronuclear envelope breakdown and chromosome condensation with three groups of haploid genomes (arrowheads)
  • d removal of the triploid mitotic genome, e-g, clones at the 4-cell, morula and blastocyst stage derived after CT of an ES cell genome into a polyspermic zygote
  • h diagram of fertilization with a failure to extrude the second polar body
  • Figure 6 shows one embodiment of reprogramming activity of zygotes enucleated in interphase or 'enucleated' in mitosis. Shown is the developmental potential of cloned embryos with donor nuclei/genomes of different developmental stages, a-d, NT into zygotes enucleated in interphase, e-h, transfer of genomes in mitosis into mitotic zygotes that had their chromosomes previously removed. Note the large placental size of ES cell clones (h), but not of zygote clones or any of the blastomere clones (a, e-g).
  • FIG. 7 shows one embodiment of construction of tetO-H2B-cherry ES cells, a, b CoIAI locus of KH2 cells before (a) and after (b) Flp-in of the H2B-cherry transgene. These cells also harbor the reverse tet-transactivator allowing doxycycline inducible regulation of the integrated transgene. c, These H2b-Cherry transgenic mouse produced from these cells, 5 days after addition of doxycycline to the drinking water and a non- transgenic control mouse observed under red fluorescence.
  • FIG 8 shows one embodiment of nuclear reprogramming after somatic cell chromosome transfer (SCCT) into zygotes in mitosis
  • SCCT somatic cell chromosome transfer
  • a Diagram of SCCT into zygotes and nuclear reprogramming.
  • Oct4::GFP transgenic tail-tip cells were arrested in mitosis and then injected into mitotic zygotes. After release from cell-cycle arrest, the embryos routinely cleaved and developed to the morula and blastocyst stages. Reprogramming of the somatic genome leads to the expression of Oct4::GFP.
  • b Culture of tail-tip cells isolated from an adult mouse carrying an Oct4::GFP transgene, phase contrast and green fluorescence.
  • Oct4::GFP SCCT embryo Note the complete absence of transgene expression in this cell culture, c-f, development of an Oct4::GFP SCCT embryo. Hours indicate the time after NT and MG- 132 release, c, 2-cell stage and 4-cell stage SCCT embryos, d, 6-cell stage SCCT embryo. Expression of Oct4::GFP first became detectable, e, SCCT embryo at the morula stage with strong expression of the GFP transgene. f, SCCT blastocyst with Oct4::GFP expression.
  • Figure 9 shows one embodiment of re-derivation of chromosome transfer (CT) ES cells from zygote CT ES cell blastocysts.
  • A Outgrowth of an inner cell mass from a zygote ES CT blastocyst on irradiated mouse embryonic fibroblast (MEF) feeders.
  • C Mitotic chromosomal spread of a zygote CT ES cell line with the complete set of 40 mouse chromosomes
  • D a 100% agouti adult male chimera that also transmitted the ES cell transgene to its offspring.
  • Figure 10 shows one embodiment of an alternative method for arresting zygotes in mitosis with a spindle using only low nocodazole concentrations, a, Zygote in prophase of mitosis 30 minutes after being transferred from O.l ⁇ g/ml nocodazole into KSOM for 5 minutes and then into 0.03 ⁇ g/ml nocodazole on the heated stage of the microscope.
  • Note the dispersed chromosomes only partially assembled into a metaphase plate and compare to Fig. ID where the chromosomes form a tight, distinct metaphase plate visible 30 min after the complete removal of nocodazole from the media.
  • low nocodazole concentrations allowed sufficient spindle polymerization to enable location of the chromosomes by HMC.
  • b The same zygote after the removal of the spindle chromosome complex.
  • Figure 11 shows one embodiment of cell cycle synchronization and reprogramming after nuclear transfer of interphase cells into mitotic zygotes a. Nuclei of fibroblasts in interphase are transferred into a zygote in mitosis that had its own genome removed. Chromosomes are marked with the red fluorescent fusion protein H2B-cherry. After release from MG- 132 mitotic arrest, chromosomes segregate randomly into two groups and form two nuclei within the same cell due to the inhibition of cytokinesis by cytochalasin B. b. - e. Mitotic progression of nuclear transfer clones. Time indicates the time after nuclear transfer, g-j.
  • Figure 12 shows development and reprogramming after mitotic genome transfer into interphase zygotes
  • a Diagram of transfer of a mitotic ES cell genome (red) into an H2B-GFP positive (green) zygote in interphase
  • b Zygote immediately after transfer
  • c zygote 2h after transfer stained for laminB marking a nuclear envelope of the zygotic interphase nuclei but not around the injected mitotic genome from the ES cell.
  • zygote in mitosis in the presence of nocodazole A difference in intensity of green and red histones on the two zygotic (haploid maternal and haploid paternal) and the diploid ES cell genome was observed, e.
  • ES cell line derived from a cloned blastocyst expressing H2B-cherry but not H2B-GFP.
  • j diploid chromosome complement of the ES cell line.
  • k chimeric mouse with mostly agouti coat color marking ES cell origin, l-o.
  • Injection of the ES cell genome without removal of the zygotic genome results in the development of clones expressing both H2B-cherry and H2B-GFP.
  • 1. absence of nocodazole results in the assembly of both ES cell genome and zygotic genomes in a single spindle, m-o.
  • FIG 13 shows developmental outcome influenced by the removal and transfer of developmental factors a.
  • recipient cytoplasts are generated either by complete enucleation in interphase, by breaking the nuclear envelope prior to removal of the interphase chromatin or by extraction of mitotic chromosomes.
  • Each of these cytoplasts is transferred with either an entire mitotic ES cell b, a mitotic 8-cell stage karyoplast c, a mitotic zygotic karyoplast, an 8-cell stage interphase nucleus (e) or an interphase nucleus of a zygote (f).
  • Figure 14 shows that Brg-1 is associated with chromatin in interphase zygotes and excluded in mitosis, a. a zygote in interphase with intact paternal and maternal pronuclei, b. zygote nuclei removed from an interphase zygote. Nuclei from several zygotes are shown in this image, c. 2-cell stage clone after enucleation in interphase and transfer of a mitotic ES cell genome, d. 2-cell stage un-manipulated control embryo, e. M-phase zygote arrested with nocodazole. f. cytoplast and karyoplast (arrow) of an M-phase zygote arrested in nocodazole. g.
  • 2-cell stage clone after genome removal in mitosis and transfer of a mitotic ES cell genome h. a zygote in interphase with a nucleus broken by micromanipulation (star*) and an intact pronucleus.
  • Brg-1 was observed on the chromatin even in the broken nucleus, albeit at a lower intensity than of the intact nucleus.
  • Some of the green speckles in the cytoplasm may have been staining artefacts, i. nuclei of blastomeres of a cleavage stage embryo in interphase and in mitosis (arrow). Brg-1 was absent from mitotic chromatin.
  • Figure 15 shows one embodiment of transfer of mitotic genomes into zygotes in anaphase, a. a zygote 50 min. after release from nocodazole block, b. karyoplast removed from a zygote in anaphase. Two groups of chromosomes were observed, c. anaphase cytoplast. The elongation seen in a. completely regressed, d. cytoplast of a cleaving zygote 24 h after genome removal. Unlike a cytoplast generated in prometaphase, an anaphase cytoplast does not attempt cleavage. e,f. anaphase cytoplast transferred with a somatic interphase nucleus.
  • a somatic donor cell before transfer g. anaphase cytoplast transferred with an M-phase genome
  • k. in anaphase Both 2Oh post transfer.
  • Figure 16 shows one embodiment of nuclear structure after enucleation of zygotes in interphase, a. specific removal of the chromatin from an interphase nucleus.
  • the transfer of a mitotic genome results in cleavage and a morphologically normal 2-cell stage embryo. Arrowheads point to nucleoli, b. Removal of intact nuclei from interphase zygotes. Transfer of a mitotic genome results in cleavage.
  • Daughter cells have small nuclei without the prominent nucleoli typical of a cleavage stage embryo, c. unmanipulated control embryo. Arrows point to nucleoli.
  • Figure 17 shows one embodiment of arrest of zygotes in mitosis with a spindle by low nocodazole concentrations.
  • Mouse zygotes were arrested in mitosis with 0.02 ug/ml nocodazole.
  • a. The granule-free area where the spindle is located is circled
  • b. Hoechst 33342 DNA staining. Chromosomes are highly unorganized, typical of early prometaphase, c. merge, d. microtubule birefringence. Regular microtubule structures that cause birefringence are basically absent (compare with h).
  • e-g. removal of the spindle also removes all chromosomes, h.
  • FIG 18 shows one embodiment of genome-exchange of blastomeres in mitosis.
  • Blastomeres were arrested in O.l ⁇ g/ml nocodazole.
  • the genome of mouse blastomeres was then removed in mitosis.
  • the mitotic genome of a different cell was introduced.
  • the genome of the donor cell was marked with H2B-cherry to follow the transferred (red) genome.
  • the blastomeres then cleaved to 2 cells, that have red interphase nuclei with the donor cell genome. These cells then contributed to the developing embryo to form a blastocysts (blast).
  • Figure 19 shows one embodiment of stem cell derivation from blastomeres transferred in mitosis.
  • Figure 20 shows one embodiment of reprogramming the genome of a somatic cell by blastomeres.
  • Figure 21 shows one embodiment of somatic cell nuclear transfer (SCNT) in human cells.
  • Donor cell refers to a cell containing nuclear-derived genetic material that is to be introduced into a recipient cell.
  • a donor cell comprises a somatic cell.
  • a donor cell may be a somatic cell such as: a neural cell, a glial cell, an astrocyte, a muscle cell, a fibroblast cell, an epithelial cell, an epidermal cell, an immune cell, a dendritic cell, a keratinocyte, an adipose cell, a chondrocyte, a cumulus cell, an esophageal cell, a melanocyte, a hematopoietic cell, a monocyte, or a mononuclear cell.
  • somatic cell such as: a neural cell, a glial cell, an astrocyte, a muscle cell, a fibroblast cell, an epithelial cell, an epidermal cell, an immune cell, a dendritic cell, a keratinocyte, an
  • a donor cell comprises a stem cell.
  • a donor cell may be an embryonic stem cell, an adult stem cell, or an umbilical cord stem cell.
  • stem cells may be used as a donor cell and will be aware of appropriate methods of isolating nuclear-derived genetic material from such cells.
  • nuclear-derived genetic material from a donor cell is introduced into a recipient cell by injection.
  • nuclear-derived genetic material from a donor cell is introduced into a recipient cell by fusion of a donor cell containing nuclear-derived genetic material with a recipient cell that lacks nuclear- derived genetic material.
  • One of ordinary skill in the art will be aware of other suitable methods for introducing nuclear-derived genetic material from a donor cell into a recipient cell.
  • Embryonic stem cell The terms “embryonic stem cell” and “ES cell” as used herein refer to an undifferentiated stem cell that is derived from the inner cell mass of a blastocyst embryo and is pluripotent, thus possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo. Embryonic stem cells appear to be capable of proliferating indefinitely, and of differentiating into all of the specialized cell types of a mammal, including the three embryonic germ layers (endoderm, mesoderm, and ectoderm), and all somatic cell lineages and the germ line.
  • embryonic stem cells have been shown to be capable of being induced to differentiate into cardiomyocytes (Paquin et al., Proc. Nat. Acad. ScL, 99:9550-9555, 2002), hematopoietic cells (Weiss et al., Hematol. Oncol. Clin. N. Amer., 11(6): 1185-98, 1997; also U.S. Pat. No.
  • Mitosis The term “mitosis” as used herein refers to the process in cell division by which the nucleus divides, resulting in two new nuclei, each of which contains a complete copy of the parental chromosomes. During mitosis, the nuclear envelope breaks down and chromosome pairs condense and attach to fibers that pull the sister chromatids to opposite sides of the cell in preparation for cytokinesis. As will be understood by those of ordinary skill in the art, mitosis is one phase of the cell cycle and is distinguished from the much longer interphase, where the cell prepares itself for division. Mitosis is typically described as consisting of four stages: prophase, metaphase, anaphase, and telophase.
  • nuclear-derived genetic material refers to genetic material that is typically sequestered in the nucleus during the interphase portion of the cell cycle. As will be understood by those of ordinary skill in the art, in the case of eukaryotic cells, such nuclear-derived genetic material includes the cell's nuclear DNA complement. In certain embodiments, nuclear- derived genetic material that is isolated from a donor or precursor cell will also include non-nucleic acid components that are beneficial and/or necessary for the proper function of the cell's nuclear DNA.
  • nuclear-derived genetic material isolated from a donor or precursor cell may also comprise histone and other chromatin proteins, proteins that are associated with or bound to the cell's nuclear DNA, proteins that function in DNA replication and/or transcription, RNA including, but not limited to, rRNA, tRNA, mRNA, and/or snRNA, lipids, small molecules and/or other components that are beneficial and/or necessary for the proper function of the cell's nuclear DNA.
  • RNA including, but not limited to, rRNA, tRNA, mRNA, and/or snRNA
  • lipids lipids
  • small molecules and/or other components that are beneficial and/or necessary for the proper function of the cell's nuclear DNA.
  • mitochondria contain genetic material in the form of DNA that is passed down from cell to cell during the process of cell division. Due to current technological limitations, removal of nuclear- derived genetic material from a donor cell may also result in the removal of mitochondria and/or other genetic material that is not nuclear-derived, which non-nuclear derived genetic material may subsequently be introduced into a recipient cell. Such extraneous non-nuclear derived genetic material is not considered to be nuclear-derived genetic material for the purposes of the present disclosure, and is not expected to have any significant effects on inventive transgenic cells disclosed herein. [0041] In certain embodiments, nuclear-derived genetic material is removed by arresting a donor or precursor cell in mitosis and removing the chromatin.
  • a donor or precursor cell is arrested in mitosis such that the nuclear- derived genetic material has condensed, thus making it easier to identify and remove.
  • a donor or precursor cell may be arrested in metaphase such that the condensed chromosomes are lined up on the metaphase plate. Removal of such lined-up condensed chromosomes is within the ordinary skill of those in the art.
  • nuclear-derived genetic material is isolated removing an intact nucleus from a donor cell.
  • nuclear-derived genetic material introduced into a recipient cell is sufficient to reprogram the recipient cell and/or drive the recipient cell through the remainder of mitosis.
  • a recipient cell into which nuclear-derived genetic material from a donor cell has been introduced is capable of dividing and giving rise to a transgenic animal.
  • a recipient cell into which nuclear-derived genetic material from a donor cell has been introduced may be manipulated to generate an embryonic stem cell.
  • Precursor cell refers to a cell containing nuclear-derived genetic material that is to be removed to generate a recipient cell.
  • a precursor cell comprises a fertilized zygote.
  • a precursor cell comprises a blastomere.
  • nuclear-derived genetic material is preferably removed from the precursor cell during mitosis.
  • a precursor cell is arrested in mitosis, such that the nuclear envelope has at least partially broken down, before the nuclear-derived genetic material is removed.
  • a precursor cell may be arrested in metaphase, thus permitting easy identification and removal of condensed chromosomes lined up on the metaphase plate.
  • Recipient cell refers to a cell derived from a precursor cell from which nuclear-derived genetic material has been removed.
  • a recipient cell is derived from a fertilized zygote.
  • a recipient cell is derived from a blastomere.
  • a recipient cell is arrested in mitosis.
  • a precursor cell may be arrested in mitosis and the nuclear-derived genetic material removed, resulting in a recipient cell that is arrested in mitosis.
  • a recipient cell of the present invention is capable of receiving nuclear-derived genetic material from a donor cell, generating a transgenic cell.
  • such a transgenic cell is capable of proceeding through mitotic and/or embryonic development.
  • Somatic cell nuclear transfer generally refers to the process of transferring nuclear material from a somatic donor cell to another cell.
  • transferred nuclear material is in the form of condensed chromosomes isolated from a somatic donor cell.
  • transferred nuclear material is in the form of intact nuclei isolated from a somatic donor cell.
  • somatic cell nuclear transfer is typically understood in the art to be limited to transfer of nuclei into oocytes, as will be clear from this disclosure, the present invention encompasses the finding that somatic cell nuclear transfer may be achieved by introducing nuclear-derived genetic material from a somatic donor cell into a recipient cell derived from a fertilized zygote or blastomere.
  • methods disclosed herein expand the usefulness of somatic cell nuclear transfer by permitting transfer into recipient cells that are derived from cells other than oocytes, methods of the present invention are not limited to the transfer of nuclear-derived genetic material isolated from somatic cells.
  • methods of the present invention are useful in the transfer of nuclear-derived genetic material isolated from non-somatic cells, including but not limited to, stem cells and/or embryonic cells.
  • stem-cell producing condition The term “stem-cell producing condition” as used herein refers to a condition or set of conditions that permits and/or drives a cell to become a stem cell.
  • an embryonic cell is permitted and/or driven to become an embryonic stem cell by subjecting such an embryonic cell to a stem- cell producing condition.
  • an embryonic blastomere may permitted and/or driven to become an embryonic stem cell by isolating the embryonic blastomere from the inner cell mass of a blastocyst and culturing the embryonic blastomere under stem-cell producing conditions, such that at least one blastomere proliferates into a pluripotent embryonic stem cell.
  • a transgenic embryonic stem cell is generated by producing a transgenic cell according to one or more methods of the present invention, allowing the transgenic cell to develop into a transgenic blastocyst comprising a plurality of transgenic blastomeres, isolating one or more transgenic blastomeres from the inner cell mass of the transgenic blastocyst, and culturing the isolated transgenic blastomere(s) under stem-cell producing conditions such that at least one transgenic blastomere develops into a pluripotent transgenic embryonic stem cell.
  • Stem-cell producing conditions can vary between species.
  • leukemia inhibitory factor is necessary and sufficient to prevent differentiation of mouse embryonic stem cells and to allow them to grow in an undifferentiated state indefinitely.
  • LIF leukemia inhibitory factor
  • primate embryonic stem cells at least one group has reported that growth on a fibroblast feeder layer is required to prevent them from differentiating (see e.g., US Patent numbers 5,843,780 and 6,200,806, incorporated herein by reference in their entirety).
  • stem-cell producing conditions including, but not limited to, culture media and/or culturing conditions that permit and/or drive a cell of a given species to become a stem cell.
  • the present invention provides transgenic cells comprising a mitotically-arrested fertilized zygote or blastomere from which endogenous nuclear-derived genetic material has been removed, and into which exogenous nuclear- derived genetic material has been introduced.
  • the present invention encompasses the discovery that such transgenic cells are capable of reprogramming exogenous introduced nuclear-derived genetic material, such that the transgenic cell is capable of undergoing mitosis and permitting the cell to be used in various therapeutic applications including, but not limited to, embryonic stem cell generation, disease modeling, drug screening and/or therapeutic cloning.
  • ES cells Reprogramming factors in ES cells (Tada, M., et al., Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells, Embo J 16, 6510-20, 1997; Tada, M., et al., Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells, Curr Biol 11, 1553-8, 2001; Cowan, C.
  • mice cloned from ES cell nuclei by chromosome transfer into zygotes displayed at least two phenotypes commonly observed in cloned animals: neonatal respiratory failure and placental overgrowth (see Example 5; also, Eggan, K. et al., 2001). Therefore, these phenotypes, common to many cloned animals, do not result solely from artificial oocyte activation.
  • mice derived by transferring chromosomes from blastomeres into zygotes did not display these phenotypes, suggesting that they do not arise solely from this mechanical procedure but likely result from failures in nuclear reprogramming.
  • the present disclosure demonstrate that reprogramming activities are not permanently lost from the egg cytoplasm following fertilization, which is relevant to the ongoing efforts to produce human ES cell lines by somatic cell nuclear transfer.
  • human oocytes are aged ones that failed to be fertilized during in vitro fertilization reactions.
  • Mouse oocytes aged in this way have reduced developmental competence and their human counterparts have thus far not been suitable recipients for nuclear transfer.
  • fresh unfertilized human oocytes would be preferable, there are substantial logistical, medical and societal difficulties in obtaining sufficient numbers.
  • normal fertilized zygotes are frozen with some regularity .
  • Such aneuploid zygotes which are estimated to number in the tens of thousands each year in the United States (Assisted reproductive technology in the United States: 2000 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry, Fertil Steril 81, 1207- 20, 2004), contain more than two pronuclei and are systematically discarded. Such zygotes are excluded from clinical use at the 1-cell stage because their abnormal ploidy is incompatible with normal postimplantation development and they could be donated for research without interfering with a couple's reproductive efforts.
  • Human polyspermic zygotes routinely undergo cleavage division and might therefore be arrested in mitosis and the zygotic chromosomes removed to generate a recipient cell according to one or more methods of the present invention.
  • the reconstructed zygotes after introducing the correct number of human somatic chromosomes from a donor cell into such a recipient cell, the reconstructed zygotes are capable of supporting development such that it is capable of being used in various therapeutic applications including, but not limited to, embryonic stem cell generation, disease modeling, drug screening and/or therapeutic cloning.
  • methods and compositions of the present invention provide novel and technically feasible avenues towards the production of "genetically tailored" human ES cell lines that are not constrained by the traditional limitations of oocyte donation for research.
  • a recipient cell is generated by removing nuclear-derived genetic material from an embryonic blastomere (e.g., from a 2, 4, 8 or more cell embryo). Nuclear-derived genetic material from a donor cell may be introduced into such a recipient cell using one or more methods described herein.
  • a recipient cell is generated by removing nuclear-derived genetic material from an ES cell that has been arrested in mitosis. Use of mitotically-arrested discarded excess human preimplantation embryos and embryonic stem cells as precursor cells to generate recipient cells that can reprogram nuclear-derived genetic material from a donor cell would substantially advance efforts to produce human ES cell lines for disease modeling and transplantation medicine.
  • a recipient cell is generated by arresting a precursor cell is arrested in mitosis and removing nuclear-derived genetic material.
  • nuclear envelope breakdown during mitosis releases one or more factors that permit the cell to reprogram nuclear-derived genetic material derived from a donor cell.
  • the present invention encompasses embodiments wherein a precursor cell is arrested at any stage of mitosis after the nucleus has begun, at least partially, to break down.
  • a precursor cell is arrested in the metaphase stage of mitosis.
  • Metaphase is characterized by condensed chromosomes that are aligned on the metaphase plate, permitting relatively easy identification and removal of the condensed chromosomes.
  • the present invention is not limited to use of recipient cells derived from precursor cells arrested in the metaphase of mitosis.
  • a recipient cell is derived from a precursor cell that has been arrested in another stage of mitosis, such as, for example, prophase, anaphase or telophase.
  • one or more compounds are used to arrest a precursor cell in mitosis prior to removing nuclear-derived genetic material.
  • One of ordinary skill in the art will be aware of compounds that halt mitosis (e.g., immediately or shortly after subjecting a cell to such a compound) and/or prevent a cell from proceeding past a certain stage of mitosis.
  • a proteasome inhibitor is used to arrest a cell in mitosis.
  • the proteasome inhibitor MG- 132 has been shown to prevent degradation of certain proteins comprising the proteasome, including the cyclinB subunit of the maturation promotion factor. A cell subjected to MG-132 is prevented from proceeding past metaphase.
  • a precursor cell is subjected to MG-132 and the nuclear-derived genetic material (in the form of condensed chromosomes lined up on the metaphase plate) is removed to generate a recipient cell.
  • the nuclear-derived genetic material in the form of condensed chromosomes lined up on the metaphase plate
  • long term exposure to MG-132 compromises proper development of a cell, even after the MG-132 has been removed.
  • Non-limiting exemplary proteasome inhibitors include peptide aldehydes (such as MG 115, and PSI), lactacystin, epoxomicin, YU-IOl and bortezomib.
  • proteasome inhibitors that halt mitosis and/or prevent a cell from proceeding past a certain stage of mitosis that can be used in accordance with the present invention.
  • non- proteasome inhibitor compounds that halt mitosis and/or prevent a cell from proceeding past a certain stage of mitosis that can be used in accordance with the present invention.
  • Non- limiting examples of non-proteasome inhibitor compounds that halt mitosis and/or prevent a cell from proceeding past a certain stage of mitosis include nocodazole, Vinca alkaloids, colchicine, vinblastine (VB), vincristine, and podophyllotoxin.
  • the microtubule depolymerizing drug nocodazole has been shown to disrupt assembly of a correctly formed spindle, which is required for proper chromosome segregation during mitosis.
  • a precursor cell is subjected to nocodazole and nuclear-derived genetic material is removed to generate a recipient cell.
  • the present invention encompasses methods of generating transgenic cells comprising subjecting a precursor cell to any compound that arrests a cell in mitosis as long as the nuclear-derived genetic material is capable of being removed to generate a recipient cell.
  • a recipient cell is generated by subjecting a precursor cell to two or more compounds that halt mitosis (e.g., immediately or shortly after subjecting a cell to such a compound) and/or prevent the precursor cell from proceeding past a certain stage of mitosis.
  • a precursor cell is subjected to two or more compounds simultaneously.
  • two compounds may be used simultaneously at concentrations wherein neither compound alone would arrest a precursor cell in mitosis, but wherein the combination of the two compounds together functions to arrest the cell in mitosis.
  • a precursor cell is subjected to two or more compounds sequentially.
  • a precursor cell may be subjected to a first compound for a short period of time, after which the precursor cell is removed from the first compound is then subjected to a second compound.
  • Such embodiments are advantageous where one or both compounds compromise proper development of a cell after exposure (e.g., as in the case of MG-132) and/or are otherwise detrimental to the precursor cell and/or the process of generating a recipient cell.
  • a precursor cell is first treated with a compound that results in chromosome condensation, and is then treated with a compound that permits easy identification and/or removal of the condensed chromosomes.
  • compounds can be advantageously administered sequentially to a precursor cell and will be able to utilize such compounds in accordance with certain inventive methods disclosed herein.
  • nuclear-derived genetic material is removed from a donor cell through the use of a pipette or other needle-like apparatus.
  • a pipette or other needle-like apparatus Such techniques and apparatuses are known to those skilled in the art and have previously been used in the context of removing nuclei for use in SCNT into activated oocytes.
  • one technique is to stain the DNA with Hoechst 33342 or other suitable dye, and then to expose the stained DNA to ultra-violet light to determine the location of the stained DNA, prior to extraction.
  • the spindle body is then aspirated according to epifluorescence imaging.
  • Another technique is to use a large enucleation needle to aspirate the first polar body and the cytoplasm under the polar body, and the
  • Karyoplast is then stained with Hoechst 33342 or other suitable dye, and checked under
  • a donor cell is a somatic cell.
  • a donor cell may be a neural cell, a glial cell, an astrocyte, a muscle cell, a fibroblast cell, an epithelial cell, an epidermal cell, an immune cell, a dendritic cell, a keratinocyte, an adipose cell, a chondrocyte, a cumulus cell, an esophageal cell, a melanocyte, a hematopoietic cell, a monocyte, or a mononuclear cell.
  • Methods of the present invention extend the utility and applicability of SCNT by permitting transfer of nuclear material derived from a somatic cell into a recipient cell derived from a fertilized zygote or blastomere, as opposed to an oocyte.
  • a donor cell is a stem cell.
  • a donor cell may be an embryonic stem cell, an adult stem cell, or an umbilical cord stem cell.
  • a recipient cell generated from a fertilized zygote or a blastomere is capable of reprogramming nuclear- derived genetic material removed from a somatic or stem donor cell.
  • nuclear-derived genetic material is removed after arresting a donor cell in mitosis.
  • a donor cell is arrested in mitosis such that the nuclear-derived genetic material has condensed, thus making it easier to identify and remove.
  • a donor cell may be arrested in metaphase such that the condensed chromosomes are lined up on the metaphase plate. Removal of such lined-up condensed chromosomes is within the ordinary skill of those in the art.
  • a donor cell is arrested in mitosis at the same stage as the precursor cell into which nuclear-derived genetic material is to be introduced.
  • a donor cell is arrested in mitosis by the same method used to arrest the precursor cell into which nuclear-derived genetic material is to be introduced.
  • a donor cell and precursor cell may be arrested in mitosis by subjected the cells to the same compound or compounds.
  • a donor cell is arrested in mitosis at a different stage as the precursor cell into which nuclear-derived genetic material is to be introduced.
  • a donor cell is arrested in mitosis by a different method that the method used to arrest the precursor cell into which nuclear- derived genetic material is to be introduced.
  • a donor cell and precursor cell may be arrested in mitosis by subjected the cells to a different compound or compounds.
  • a donor cell is not arrested in mitosis.
  • nuclear-derived genetic material is isolated removing an intact nucleus from a donor cell in interphase.
  • somatic or stem cells may be more or less susceptible to different methods of removing their nuclear-derived genetic material.
  • the precise method of removing nuclear-derived genetic material from a donor cell is not critical so long as the nuclear-derived genetic material removed from the donor cell is capable of being transplanted into a recipient cell such that the resulting transgenic cell is capable of proceeding through mitosis and/or the beginning stages of development.
  • One of ordinary skill in the art will be aware of other suitable methods for removing such nuclear-derived genetic material from a donor cell, as well as suitable conditions for culturing a donor cell prior to, during and after removal of the nuclear-derived genetic material.
  • nuclear-derived genetic material removed from a donor cell is introduced into a recipient cell via injection.
  • Such techniques are known to those skilled in the art and have previously been used in the context of SCNT into activated oocytes.
  • the present invention encompasses the discovery that that nuclear transfer, including but not limited to somatic cell nuclear transfer, may be achieved by introducing nuclear-derived genetic material from a somatic donor cell into a recipient cell derived from a fertilized zygote or blastomere.
  • methods of the present invention are useful in the transfer of nuclear-derived genetic material from a somatic donor cell into a recipient cell, such that the somatic nuclear-derived genetic material is reprogrammed and becomes capable of supporting
  • nuclear-derived genetic material extracted from a donor cell is introduced into a recipient cell via electroporation.
  • nuclear- derived genetic material can be extracted from a donor cell arrested in mitosis in the form of condensed chromosomes.
  • Such condensed chromosomes can be introduced into a recipient cell by incubating the recipient cell with the condensed chromosomes, and subjected the incubated mixture to an electric field sufficient in strength to perforate the plasma membrane of the recipient cell, permitting the condensed chromosomes to enter the cell.
  • nuclear-derived genetic material can be removed from a donor cell arrested in mitosis in the form of an intact nucleus.
  • nuclear-derived genetic material is introduced into a recipient cell by fusion of a donor cell containing the nuclear-derived genetic material with a recipient cell.
  • a donor cell may be fused with a recipient cell by immersing the donor and recipient cells into solution containing a chemical that promotes cell fusion (e.g., 50% PEG 1500 and/or other suitable surfactant).
  • a donor cell may be fused with a recipient cell by employing an external electric field (e.g., using methods developed for hybridoma formation and/or derivatives thereof), resulting in cell perforation, after which the perforated cells fuse with each other.
  • a donor cell may be fused with a recipient cell by subjecting the cells to a focused laser beam that punctures holes in the cell membranes, after which the fenestrated cells fuse with each other. Laser induced cell fusion typically takes place under microscopic observation and easily enables the study of the fusion dynamics.
  • One of ordinary skill in the art will be aware of other suitable techniques for fusing a donor cell containing nuclear-derived genetic material with a recipient cell.
  • the precise method of introducing nuclear-derived genetic material from a donor cell into a recipient cell is not critical so long as the recipient cell is capable of accepting nuclear-derived genetic material removed from the donor, such that the resulting transgenic cell is capable of proceeding through mitosis and/or the beginning stages of development.
  • One of ordinary skill in the art will be aware of other suitable methods for introducing nuclear-derived genetic material from a donor cell into a recipient cell.
  • suitable conditions for culturing a recipient cell prior to and during transplantation as well as suitable conditions for culturing a transgenic cell and after transfer of the nuclear-derived genetic material.
  • nuclear-derived genetic material of a donor cell is introduced into a recipient cell after genetic material of the recipient cell has been removed.
  • nuclear-derived genetic material of a donor cell is introduced into a recipient cell before genetic material of the recipient cell is removed.
  • nuclear-derived genetic material of a donor cell may be introduced into a recipient cell (e.g., a recipient cell in interphase), and genetic material of the recipient cell is subsequently removed (e.g., when the recipient cell is arrested in mitosis).
  • the recipient cell is treated with one or more agents that inhibit cleavage or stimulate mitotic exit without cleavage.
  • the cell is treated with cytocholasin B, or with an agent that inhibits a mitotic kinase.
  • methods of the present invention may be advantageously employed to generate a stem cell including, but not limited to, an embryonic stem cell.
  • Stem cells typically share two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that are capable of maintaining their unspecialized state and of renewing themselves for long periods through cell division. Second, under appropriate conditions, they can be induced to differentiate into cells with specialized functions. Several types of stem cells have been identified including adult stem cells, umbilical cord stem cells, and embryonic stem cells.
  • Embryonic stem cells may be characterized by several criteria, which will be known by those of ordinary skill in the art. For example, embryonic stem cells are typically capable of continuous indefinite replication in vitro. Continued proliferation for a long period of time (e.g., 6 months, one year or longer) of culture is a sufficient evidence for immortality, as primary cell cultures without this property fail to continuously divide for such a length of time (Freshney, Culture of animal cells. New York: Wiley-Liss, 1994). In certain embodiments, embryonic stem will continue to proliferate in vitro under appropriate culture conditions for longer than one year, and maintain the developmental potential to contribute all three embryonic germ layers throughout this time.
  • Such developmental potential can be demonstrated by the injection of embryonic stem cells that have been cultured for a prolonged period (over a year) into SCID mice and then histologically examining the resulting tumors.
  • length of time in culture is not the sole criteria that may be used to identify an embryonic stem cell, and even though cells have grown in culture for less than 6 months, such cells may nevertheless be embryonic stem cells.
  • embryonic stem cells may be identified by the expression of certain markers, including but not limited to cell surface markers.
  • embryonic stem cells from different species will exhibit species-specific markers on their cell surfaces.
  • Thomson US Patent Numbers 5,843,780 and 6,200,806, each of which is incorporated herein in its entirety by reference
  • SSEAs Stage Specific Embryonic Antigens
  • Embryonic stem cells derived from different species exhibit different patterns of SSEAs.
  • undifferentiated primate ES cells include human ES cells express SSEA-3 and SSEA-4, but not SSEA-I.
  • undifferentiated mouse ES cells express SSEA-I, but not SSEA-3 or SSEA-4.
  • markers that are not exhibited on the surface of a cell may be used to identify an embryonic stem cell.
  • the homeodomain transcription factor Oct 4 also termed Oct-3 or Oct3/4 is frequently used as a marker for totipotent embryonic stem cells.
  • embryonic stem cells may be identified by the capacity to develop into all of the specialized cell types of a mammal, including the three embryonic germ layers (endoderm, mesoderm, and ectoderm), and all somatic cell lineages and the germ line. Additionally and/or alternatively, embryonic stem cells may be identified by the capacity to participate in normal development when transplanted into a preimplantation embryo to generate a chimeric embryo.
  • a transgenic cell is generated by transferring nuclear-derived genetic material from a donor cell to a recipient cell, after which the transgenic cell is allowed to develop into a blastocyst and a blastomere cell from the inner cell mass is isolated and/or cultured (and optionally passaged for several generations) under stem-cell producing conditions, resulting in generation of an embryonic stem cell syngenic with the nuclear- derived genetic material removed from the donor cell used to generate the transgenic cell.
  • Methods of the present invention are employed to generate a human stem cell including, but not limited to, a human embryonic stem cell.
  • methods of the present invention are employed to generate a non-human stem cell including, but not limited to, a non-human embryonic stem cell.
  • cells from the inner cell mass are cultured in a culture dish that is coated with a feeder layer comprising mouse embryonic skin cells that have been treated so they will not divide.
  • a feeder layer gives the inner cell mass cells a sticky surface to which they can attach and also releases nutrients into the culture medium.
  • cells from the inner cell mass are cultured in a culture dish that is not coated with a feeder layer.
  • Stem-cell producing conditions are known to those of ordinary skill in the art and can often vary between species.
  • leukemia inhibitory factor LIF
  • LIF leukemia inhibitory factor
  • primate embryonic stem cells at least one group has reported that growth on a fibroblast feeder layer is required to prevent them from differentiating (see e.g., US Patent numbers 5,843,780 and 6,200,806, incorporated herein by reference in their entirety).
  • stem-cell producing conditions including, but not limited to, culture media and/or culturing conditions that permit and/or drive a cell of a given species to become a stem cell.
  • the present invention offers great potential for developing better models for the study of human disease and/or better methods of treatment.
  • Somatic cell nuclear transfer is a powerful research tool with the potential for creating unique cell lines for studies of disease pathogenesis.
  • methods of the present invention are used to generate cell lines that contain alterations (e.g., deletions, rearrangements, duplications, substitutions, etc.) in genes associated with or thought to be associated with a particular disease.
  • nuclear-derived genetic material may be removed from a donor cell isolated from a patient exhibiting a particular disease. Such nuclear-derived genetic material may then be introduced into a recipient cell, which may then be manipulated to generate an embryonic stem cell line, that can be used to study and/or model the disease of interest.
  • a disease is modeled and/or studied by inducing an embryonic stem cell line (that has, for example, been generated by methods of the present invention to contain one or more alterations in one or more genes associated with a disease of interest) to differentiate by culturing such a cell line under appropriate differentiation conditions.
  • an embryonic stem cell line may be generated that contains one or more alterations in one or more genes associated with a neurological degenerative disease such as Alzheimer's disease.
  • Such an embryonic cell line may then be induced to differentiate into nerve cells by placing it under appropriate differentiation conditions.
  • an embryonic stem cell line that is generated to contain one or more alterations in one or more genes associated with a disease may be used to screen for agents (e.g., small molecules, biologicals, etc.) that can be used in the treatment and/or prevention of that disease.
  • agents e.g., small molecules, biologicals, etc.
  • such an embryonic stem cell line may be induced to differentiate into a cell type associated with the disease of interest by placing it under appropriate differentiation conditions.
  • the differentiating or differentiated cell may be subjected to a test agent in order to determine whether that agent has an effect on the development, progression and/or physiology of the cell, and thus potentially on disease of interest.
  • stem cell lines generated according to one or more methods of the present invention are useful in studying and/or modeling diseases that to date, have not been amenable to such study and/or modeling. For instance, in many cases, by the time a patient is diagnosed with a particular disease, the early events of disease progression and pathogenesis have already occurred, making it difficult or impossible to determine and track the molecular, cellular, or other changes that occur during the course of the disease. Using methods and compositions disclosed herein, researchers will now be able to determine and study such molecular, cellular, or other changes, leading to a better understanding of disease progression and possibly pointing the way to more effective treatments.
  • human stem cell lines are generated according to one or more methods of the present invention, which human stem cell lines are useful in studying and/or modeling diseases and/or to screen for agents (e.g., small molecules, biologicals, etc.) that can be used in the treatment and/or prevention of diseases.
  • non-human stem cell lines are generated according to one or more methods of the present invention, which non-human stem cell lines are useful in studying and/or modeling diseases and/or to screen for agents (e.g., small molecules, biologicals, etc.) that can be used in the treatment and/or prevention of diseases.
  • Non-human stem cell lines are advantageous when ethical and/or practical limitations prevent the use of human stem cell lines.
  • Non- limiting examples of non-human stem cell lines that may be generated according to one or more methods of the present invention include mouse and primate stem cell lines. Those of ordinary skill in the art will be aware of other non- human stem cell lines that will be useful and will be able to generate such stem cell lines by employing one or more methods of the present invention.
  • Methods of the present invention may be used to generate cell lines for the study and/or modeling of any number of diseases or conditions.
  • diseases or conditions include childhood congenital malformations, sickle cell anemia, neurological diseases such as amyotrophic lateral sclerosis (also known as Lou Gehrig's disease), Parkinson's disease, Alzheimer's disease or any number of other neurological diseases, Down syndrome (a condition that arises in patients with trisomy for chromosome 21 resulting in dysregulated signaling through the NFAT/calcineurin pathway), etc.
  • diseases or conditions include childhood congenital malformations, sickle cell anemia, neurological diseases such as amyotrophic lateral sclerosis (also known as Lou Gehrig's disease), Parkinson's disease, Alzheimer's disease or any number of other neurological diseases, Down syndrome (a condition that arises in patients with trisomy for chromosome 21 resulting in dysregulated signaling through the NFAT/calcineurin pathway), etc.
  • Amyotrophic lateral sclerosis also known as Lou Gehrig's
  • transgenic cells generated by one or more methods of the present invention, and/or embryonic stem cells derived from such transgenic cells comprise nuclear-derived genetic material from a somatic donor cell isolated from a particular individual.
  • Patient-specific, immune-matched human embryonic stem cells have the potential to be of great biomedical importance for studies of disease and development. For example, certain patients may respond better to a given therapy or drug regimen than other patients. Additionally or alternatively, certain patients may experience fewer and/or less severe side effects after being administered a given therapy or drug regimen than other patients.
  • embryonic stem cells containing the genetic complement of a patient suffering from a disease of interest By generating embryonic stem cells containing the genetic complement of a patient suffering from a disease of interest, and permitting such cells to differentiate into a cell type associated with that disease, it will be possible to better predict which therapy or drug regimen will be most beneficial and/or result in the least detrimental side effects.
  • patient-specific human embryonic stem cells have the potential to be of great biomedical importance for the discovery and/or development of patient-specific agents that can be used to prevent and/or treat a disease of interest.
  • embryonic stem cells containing the genetic complement of a patient suffering from a disease of interest, permitting such cells to differentiate into a cell type associated with that disease, and subjecting such differentiating or differentiated cells to one or more test agents, discovery and/or development of an agent that will be most beneficial and/or result in the least detrimental side effects for that particular patient will be facilitated.
  • One of ordinary skill in the art will be able to apply methods and compositions of the present invention to the discovery and/or development of an agent specific for a patient and/or disease of interest.
  • Embryonic stem cells generated by certain methods of the present invention are of obvious importance in the study and/or modeling of human diseases, although one of ordinary skill in the art will understand that the present disclosure is not limited to human applications.
  • non-human embryonic stem cell lines generated by certain methods of the present invention may be used in the study and/or modeling of diseases associated with pets (e.g., cats, dogs, rodents, etc.) as well as commercially important domestic animals (e.g., cows, sheep, pigs, etc.).
  • non-human embryonic stem cell lines generated by certain methods of the present invention may be used to screen for agents that can be used in the prevention and/or treatment of diseases associated with pets and/or commercially important domestic animals.
  • non-human embryonic stem cells generated by one or more methods of the present invention are used to generate a transgenic animal that may be useful in studying and/or modeling a disease of interest.
  • Non-human embryonic stem cells generated by introducing nuclear-derived genetic material from a donor cell into an oocyte have been used to generate a variety of species of cloned animals (e.g., sheep (Willadsen, 1986), rabbits (Stice & Robl, 1988), mice (Cheong et al, 1992), and other animals (Sims & First, 1994; Prather, et al., 1989; Meng et al., 1997).
  • a transgenic animal generated according to one or more methods of the present invention contains one or more alterations (e.g., deletions, rearrangements, duplications, substitutions, etc.) in genes associated with or thought to be associated with a particular disease, such that the transgenic animal exhibits a diseased state.
  • alterations e.g., deletions, rearrangements, duplications, substitutions, etc.
  • the diseased transgenic animal is capable of serving as a model to study a related disease in humans.
  • a transgenic animal may be generated that exhibits Alzheimer's disease-like symptoms.
  • such an animal is capable of serving as a model organism for the study of Alzheimer's disease in humans (e.g., disease onset, progression of the disease, phenotypes associated with the disease, drugs and/or other agents that affect the onset, progression, or phenotype of the disease, etc.).
  • Alzheimer's disease e.g., disease onset, progression of the disease, phenotypes associated with the disease, drugs and/or other agents that affect the onset, progression, or phenotype of the disease, etc.
  • a transgenic animal is generated from a single embryonic stem cell generated according to one or more methods of the present invention.
  • the nuclear material of all cells of the transgenic animal will be identical to each other and to the nuclear-derived genetic material that was originally removed from a donor cell and introduced into a recipient cell to generate a transgenic cell.
  • such transgenic animals are useful where it is desired to study and/or model global effects of a disease of interest in a homozygous animal (e.g., a disease of interest may affect more than one tissue or organ, or may result in deleterious effects that are systemic).
  • an embryonic stem cell generated according to one or more methods of the present invention is transplanted into an early-stage blastocyst in order to generate a chimeric transgenic animal.
  • the germline of a generated chimeric transgenic animal will be derived from such an embryonic stem cell, permitting the generation and isolation of germ cells (e.g., sperm, oocytes, germline stem cells) that contain nuclear-derived genetic material that was originally removed from a donor cell and introduced into a recipient cell to generate a transgenic cell.
  • germ cells e.g., sperm, oocytes, germline stem cells
  • nuclear-derived genetic material from a donor cell is altered in vitro or in vivo prior to its removal.
  • a donor cell line may be infected with a virus or other infectious agent that integrates its own genetic material into the nuclear-derived genetic material of the cell line, after which the transgenic nuclear-derived material is removed and introduced into a recipient cell.
  • a transgenic donor cell line is screened prior to removal of the transgenic nuclear-derived material to determine whether and/or where genetic material from the virus or other infectious agent has been integrated into the genome.
  • a transgenic animal generated according to one or more methods of the present invention is a mouse. In certain embodiments, a transgenic animal generated according to one or more methods of the present invention is a primate. Other non-limiting examples of transgenic animals that may be generated using methods of the present invention include cats, dogs, rodents, cows, sheep, and pigs. One of ordinary skill in the art will be aware of other transgenic animals that can be generated, and will be able to use one or more methods described herein to generate such transgenic animals.
  • transgenic cells and/or embryonic stem cell lines generated by one or more inventive methods described herein may be useful in therapeutic cloning.
  • Therapeutic cloning is a general term used to refer to the process of producing cells and/or tissues for the replacement of damaged, diseased, or lost cells and/or tissues in a patient.
  • Therapeutic cloning is distinct from reproductive cloning since the transgenic cell is not permitted to become a blastocyst that is transplanted back to the uterus.
  • Reproductive cloning is used to generate an embryo that has the identical genetic material as its cell source. Such an embryo could then be implanted into the uterus of a female to give rise to a liveborn infant that is a clone of the donor.
  • reproductive cloning has been banned in most countries for human applications.
  • therapeutic cloning is used to generate only embryonic stem cell lines whose genetic material is identical to that of its source. Such embryonic stem cells have the potential to become almost any type of cell in the adult body, and thus are useful in cell and tissue replacement applications.
  • therapeutic cloning is achieved by generating embryonic stem cells according to one or more methods described herein, after which such embryonic stem cells are driven to differentiate into a particular type of cell and/or tissue.
  • Embryonic stem cells are capable of differentiating into all of the specialized cell types of a mammal, including the three embryonic germ layers (endoderm, mesoderm, and ectoderm), and all somatic cell lineages and the germ line.
  • embryonic stem cells have been shown to be capable of being induced to differentiate into cardiomyocytes, hematopoietic cells, insulin-secreting beta cells, and neural progenitors capable of differentiating into astrocytes, oligodendrocytes, and mature neurons.
  • One of ordinary skill in the art will be aware of other cell types that have been derived from embryonic stem cells and will be aware of appropriate methods to derive such differentiated cells.
  • Such differentiated cells may be transplanted into a patient in need thereof to treat and/or reverse damage caused by, for example disease and/or injury.
  • methods of the present invention are particularly advantageous when the nuclear-derived genetic material is obtained from the cells of a patient to be treated by therapeutic cloning.
  • the resulting transgenic cells are perfectly matched to the patient's immune system, and thus are syngenic to the genome of the patient to be treated.
  • Such syngenic cells may be used to generate embryonic stem cells that are capable of giving rise to any of a variety of cell and/or tissue types that can be transplanted into the patient.
  • therapeutic cloning comprising use of such syngenic transplantable cells and/or tissues generated according to one or more of the present invention obviates the unwanted immune responses typically associated with transplantation of non-auto logous tissues and/or eliminates the requirement for administration of immunosuppressive drugs.
  • non-human cells and/or tissues derived from embryonic stem cells generated by certain methods of the present invention may be used in cell replacement therapy of pets (e.g., cats, dogs, rodents, etc.) as well as commercially important domestic animals (e.g., cows, sheep, pigs, chickens, etc.).
  • pets e.g., cats, dogs, rodents, etc.
  • commercially important domestic animals e.g., cows, sheep, pigs, chickens, etc.
  • BDFl mice used as zygote donors were obtained from Charles River laboratories and B6jcBA-Tg(Pou5fl-EGFP)2Mnn/J mice originating from the lab of Hans Schoeler (Yoshimizu, T. et al., 1999) were obtained from Jackson laboratories.
  • KH2 cells as well as plasmids pBS31tetOpgkATGfrt and pCAGGS-Flpe-pur for the construction of KH2-H2B-cherry ES cells (approx. p40) were obtained from Konrad Hochedlinger (Beard, C, et al., 2006).
  • the H2B-cherry fusion protein was constructed by recombinogenic PCR using the primers 5'- ACA CCA GCG CTA AGG ATC CAC CGG TCG CCA TGG TGA GCA AGG GCG AG-3' (SEQ ID NO: 1) and 5'- AGC CTT TAA GCC TGC CCA GAA GAC-3' (SEQ ID NO:2) and 5'- GCG ACC GGT GGA TCC TTA GCG CTG GTG TAC TTG GTG ACG GCC TTA GTA CC-3' (SEQ ID NO:3) and 5'- CAC CGT CGA CGG TAC CGC CAC CA-3' (SEQ ID NO:4) and then cloned into the EcoRI site of pBS31tetOpgkATGfrt.
  • Somatic donor cells from skin and tail were cultured as previously described (Wakayama, T. & Yanagimachi, R., Cloning of male mice from adult tail-tip cells, Nat Genet 22, 127-8, 1999).
  • doxycycline Sigma D9891 was added to the drinking water of mice at a concentration of lmg/ml and to cell cultures at l ⁇ g/ml.
  • KSOM Cemicon
  • MG- 132 Zygotes arrested in mitosis were ⁇ transferred into KSOM (Chemicon) with 1-2 ⁇ M MG- 132 for 5- 20 min and then for manipulations transferred on the stage into oil-covered droplets of HCZB supplemented with 5 ⁇ g/ml CytochalasinB (Sigma C6762) and l-2 ⁇ M MG- 132 (Calbiochem #474790).
  • MG- 132 was completely omitted and replaced with 0.0025-0.03 ⁇ g/ml nocodazole during the manipulations to delay mitotic progression of the zygote while not dissociating the spindle (Fig. 10). This modification resulted in a smaller spindle volume and a more regular cleaveage of clones.
  • Mitotic donor cells were obtained after culturing cells with 0. l ⁇ g/ml nocodazole for 6-12 hours. Cells were obtained by mitotic shake off from the culture dish, trypsinized and then mixed with 1- 7% PVP containing 0. l ⁇ g/ml nocodazole. Mitotic cells were selected under the microscope and then transferred using a lO ⁇ m needle for mitotic ES cells and 12-14 ⁇ m needle (Humagen) for mitotic somatic cells. Spindle chromosome complex removal and transfer of broken cells was done in one step, taking care that the cell was deposited approximately in the middle of the zygote. Our average survival rate was 85%.
  • Manipulation of interphase zygotes was done 24-26h post hCG with the methods described above, with the addition of 0.1-0.3 ⁇ g/ml nocodazole to the manipulation medium and the use of a 14 ⁇ m needle for enucleation and transfer.
  • Nuclei of 2-cell stage embryos were transferred by direct injection or by electro fusion using an LFlOl electro fusion apparatus with two DC pulses of 1.8kV/cm in medium containing 0.26mM mannitol, O.lmM MgSO4, 0.5mM HEPES 0.05% BSA. Images of optical birefringence were taken with the oosight system (see CRI at the website having the URL www.cri-inc.com).
  • pronuclei was scored 10 hours after the initiation of the IVF reaction and zygotes with more than 2 pronuclei were selected for chromosome transfer.
  • both the oolemma and the plasma membrane inhibit polyspermy (Wolf, J. P. et al. Absence of block to polyspermy at the human oolemma. Fertil Steril 67, 1095-102, 1997).
  • the mouse zona pellucida acts as an efficient barrier to sperm even without any fertilization.
  • chromosomes of dispermic embryos assemble in a single spindle in the center of the egg (see e.g., Kola, L, Trounson, A., Dawson, G. & Rogers, P.
  • Tripronuclear human oocytes altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod 37, 395-401, 1987).
  • cytochalasin B was added to the IVF reaction at a concentration of 3ug/ml for 5 hours.
  • a failure to extrude the second polar body occurs at a frequency of about 4% after fertilization by intracytoplasmic sperm injection (see Munne, S. & Cohen, J. Chromosome abnormalities in human embryos. Hum Reprod Update 4, 842-55, 1998).
  • mice embryonic stem cells For the derivation of mouse embryonic stem cells, cloned blastocysts were plated on irradiated mouse embryonic fibroblast feeder layers in mES medium containing the MAP kinase inhibitor PD98059 (Cell Signaling) and LIF (Chemicon). Mitotic spreads of ES cells were made by incubating ES cells for 12 hours in O.l ⁇ g/ml nocodazole to arrest them in mitosis, then they were trypsinized and incubated in 0.56% w/v KCl, stained with Hoechst and then fixed with a 1 :3 mixture of glacial acetic acid with methanol.
  • PD98059 Cell Signaling
  • LIF Cell Signaling
  • Mitotic spreads of ES cells were made by incubating ES cells for 12 hours in O.l ⁇ g/ml nocodazole to arrest them in mitosis, then they were trypsinized and incubated in
  • Chimeric mice were made by injection of ES cells into non-agouti BDF2 blastocysts and embryo transfer into d2.5 pseudopregnant albino ICR females. Cloned and control blastocysts were transferred to the uterus of d2.5 pseudopregnant ICR females. Cesaerian section was performed on day E 19.5. Surviving pups were fostered to an ICR foster mother that had given birth on either the same day or 1-4 days earlier. Primers for genotyping of puromycin, neomycin, IL-2 and TCR genes are as described by the Jackson laboratory (see the webpage having the url www.jax.org/). Experiments with animals were done in accordance with the guidelines established by the Harvard University/Faculty of Arts and Sciences IACUC for the humane care and use of animals in research.
  • Preimplantation stage embryos were stained with a Cdx2 antibody (Biogenex) as described (Strumpf, D. et al. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst, Development 132, 2093-102, 2005).
  • the secondary antibody was coupled to Rhodamine-X.
  • ES cells Fig. 5 and somatic donor cells were stained with the antibodies specific for OCT4 (Santa Cruz, sc5279) and SSEA-I (Santa Cruz, sc21702).
  • the metaphase to anaphase transition requires degradation of the cyclinB subunit of the maturation promoting factor by the proteasome (Glotzer, M., et al., Cyclin is degraded by the ubiquitin pathway, Nature 349, 132-8, 1991; Murray, A. W., Recycling the cell cycle: cyclins revisited, Cell 116, 221-34, 2004).
  • the metaphase to anaphase transition and mitotic exit are blocked (Ehrhardt, A. G. & Sluder, G., Spindle pole fragmentation due to proteasome inhibition, J Cell Physiol 204, 808-18, 2005; Table 1).
  • Zyg Zygote
  • ESC embryonic stem cell
  • 2-cell 2-cell stage embryo
  • ooc oocyte
  • Fib somatic cell fibroblast
  • M Mitosis
  • Mil metaphase of meiosis II
  • I interphase
  • manip manipulated
  • Elec electro fusion
  • Inj direct injection. # of recipient donor morulae & transferred # of
  • Example 5 Cloned mice generated by zygote chromosome transfer
  • Donor cells were arrested in mitosis with nocodazole (Fig. 2e, f) until microinjection and consequently their chromosomes were transferred into the mitotic zygotes without a spindle (Fig. 3a). Following release from MG- 132, a new spindle rapidly nucleated around the ES cell chromosomes. Chromosome segregation and cytokinesis were often observed within 90 to 150 minutes (Fig. 2g-i). Manipulated embryos cleaved at a frequency of 30% and those that cleaved developed to the morula and blastocyst stage with an efficiency of 60% (see Table 3, Fig. 3c, d).
  • Example 6 Zygotes can reprogram adult somatic chromosomes
  • Oct3/4 is expressed in early pluripotent cells but not in somatic tissues (Scholer, H. R., et al., Oct-4: a germline-specific transcription factor mapping to the mouse t-complex, Embo J 9, 2185-95, 1990 and therefore reactivation of Oct3/4 is a measure of successful reprogramming (Boiani, M., et al., Oct4 distribution and level in mouse clones: consequences for pluripotency, Genes Dev 16, 1209-19, 2002).
  • Somatic cell chromosome transfer embryos cleaved at an efficiency (30%), that was similar to that observed using ES cell chromosome donors but developed to the morula and blastocyst stage at a lower efficiency (42% of cleaved embryos, see e.g., Table 1). Consistent with successful nuclear reprogramming, Oct4::GFP expression became visible in late cleavage stage embryos and was strongly expressed at the blastocyst stage (Fig. 8).
  • Example 7 Embryonic stem cells can be derived by chromosome transfer into zygotes
  • Example 8 Re-derivation of chromosome transfer ES cells from zygote CT ES cell blastocysts
  • BDFl mice used as zygote donors were obtained from Charles River laboratories.
  • Transgenic ES expressed the H2B-cherry fusion gene under the control of the combined CMV-chicken beta actin promoter (pCAGGS-H2B-cherry). Germ-line transmission of this transgene was obtained, but mice carrying the transgene did not gain weight and all died in the second week after birth.
  • Tail tip fibroblasts used for nuclear transfer were obtained from adult B6jcBA-Tg(Pou5fl-EGFP)2Mnn/J mice.
  • Chromosome transfer in mitosis involves the removal of the host cell chromosomes including the microtubule spindle. Transfer of a new genome results in the assembly of a new spindle that is smaller than the spindle of an unmanipulated zygote. Also, clones generated this way frequently show high degrees of fragmentation. We reasoned that this may be attributed to difficulties in the assembly of a new spindle and to a reduction of microtubule material. To reduce the amount of spindle material removed, we determined a nocodazole concentration (0.02-0.03 microgram/ ml (ug/ml)) that arrests zygotes with a spindle in mitosis.
  • Such arrested zygotes have a spindle visible by light microscopy, and with a higher consistency than surrounding cytoplasm, thereby obviating the use of Hoechst and ultraviolet light to visualize the chromosomes. All chromosomes could be removed, either within a single spindle in the center of the zygote, or in two separate spindles containing paternal and maternal genomes, slightly distant from the center of the zygote. Aspirated spindle material was softer and smaller than when nocodazole was completely removed and replaced with the proteasome inhibitor MG- 132 for metaphase arrest. In the presence of MG- 132, zygotes proceed from prometaphase to metaphase within approx.
  • nocodazole arrested zygotes in early prometaphase as seen by the disorganized configuration of 21 chromosomes and a weak signal of microtubule birefringence (Figure 17a).
  • nocodazole is not toxic and embryos can be incubated in nocodazole for an additional 30 min or more after transfer. This procedure resulted in cleavage with less fragmentation and was also more convenient for the experimentator.
  • Embryos at the 8-cell stage were incubated in nocodazole for mitotic arrest at approx. 6Oh post hCG for 5 hours. Within 20-30 min after the release from the nocodazole arrest, mitotic spindles formed that could be aspirated into a needle with a 10 um diameter.
  • Tail tip fibroblasts of adult mice were cultured in 0.5% serum for 2 or more days and then transferred into zygotes arrested by nocodazole in mitosis. Subsequent to the transfer, they were further arrested by the addition of lug/ml MG-132 for 2 hours. To inhibit cytokinesis, 2.5ug/ml cytochalasinB was added to the culture medium for 6-10 hours. Alternatively, zygotes were arrested for 2 hours in 0.
  • lug/ml nocodazole after transfer and then transferred to a medium containing lOuM purvalanol A (Sigma P4484), 2mM 6-DMAP (Sigma D2629), the aurora B kinase inhibitor ZM 447439 (Ditchf ⁇ eld 2003)(Tocris Bioscience) and 0. lug/ml nocodazole for 90 to 120 minutes.
  • zygotes spontaneously escaped the nocodazole block and did not have to be exposed to purvalanol A or 6-DMAP to promote exit from mitosis. All of these methods of arrest in mitosis and inhibition of cytokinesis did result in embryos with the ability to develop to the morula and blastocyst stage.
  • mouse zygotes were arrested in prometaphase by 0. lug/ml nocodazole.
  • Zygotes arrested in mitosis were washed through one to three drops of KSOM (Chemicon) to remove residual nocodazole and then cultured in KSOM for 40-45 minutes. They were then transferred to hCZB on the heated stage of the microscope and monitored for anaphase entry.
  • Elongating zygotes were transferred to lug/ml nocodazole and 5ug/ml CytoB in hCZB, the spindle-chromosome complex was removed, and the cytoplast subsequently transferred with either a tail tip cell or an ES cell.
  • Zygotes and preimplantation stage embryos were fixed in 4% paraformaldehyde over night at 4°C, permeabilized in PBS with 0.5% Triton-XIOO (PBS/T) for 20 min., blocked in blocking solution consisting of 0.1% PBS/T with 10% FBS over night at 4°C, incubated in primary antibody at 4°C in blocking solution, then washed for Ih at room temperature in 0.1% PBS/T, incubated with secondary conjugated antibody in 0.1% PBS/T at room temperature for Ih, washed as above, stained with Hoechst 33342 for 5 minutes and used for confocal imaging.
  • PBS/T Triton-XIOO
  • Example 10 Zygotes reprogram interphase nuclei upon premature chromosome condensation
  • cytokinesis was inhibited by the addition of cytochalasinB.
  • clones did separate the condensed chromosomes into two, often unequally sized groups to form two nuclei within the same cell (Fig.11 d,e).
  • Clones proceeded to the next mitosis and cleaved to a 2-cell stage that, in terms of developmental timing, is equivalent to the 4-cell stage.
  • Development continued to both the morula and to the blastocyst stage (Fig. 1 lg-j, Table 6). Clones developing beyond the 2-cell stage strongly activated the Oct4 promoter of an Oct4-GFP transgene, showing that efficient nuclear reprogramming has accompanied preimplantation development.
  • Zygotes entered anaphase 45 min to 75 min after release from the nocodazole arrest and could be recognized by the separation of the chromosomes, the elongation of the spindle and of the cell itself (Fig. 15a). Zygotes in anaphase that were not manipulated did initiate cytokinesis and the formation of nuclei within minutes. Anaphase zygotes were chosen for removal of the genome and the removed material was stained with Hoechst33342 to verify chromosome separation (Fig. 15b). Upon removal of the anaphase spindle- chromosome complex, the elongation of the cell regressed and the cytoplast did not attempt cytokinesis for more than 24 hours (Fig. 15c, d).
  • Cytoplasts injected with a fibroblast nucleus did not undergo chromosome condensation, spindle assembly, chromosome segregation and cleavage as after nuclear transfer into a prometaphase zygote, but entered the next interphase as a single cell with a single nucleus that was initially small and of somatic morphology, but continued to enlarge within a day after transfer to display the nuclear morphology of a cleavage stage embryo (Fig. 15e, f). These clones did not proceed in development but arrested as a single cell and invariably failed to activate the Oct4-GFP transgene, even when left in culture for more than 60 hours. ES cell nuclei were then transferred in interphase into zygotes in anaphase.
  • Example 12 Reprogramming activities are present in anaphase and throughout mitosis and meiosis
  • nuclei or condensed chromosomes were transferred into 'enucleated' oocytes 60 to 90 minutes after the initiation of activation. When condensed chromosomes were transferred, development to the blastocyst stage was very efficient.
  • Example 13 Mitotic kinase activity is required after nuclear but not mitotic genome transfer
  • Example 14 Enucleation in interphase is incompatible with developmental reprogramming
  • Example 15 Zygotes in interphase are suitable recipients of a donor genome if the host genome is removed in mitosis
  • the condensed chromatin did not organize into a new nucleus as seen by the absence of laminB around the injected chromatin, and chromosomes remained condensed (Fig. 12c).
  • the injected mitotic donor cell did not alter cell cycle kinetics of the recipient.
  • These tetraploid zygotes entered the next mitosis between 30 and 34h post hCG, like unmanipulated zygotes.
  • zygotes were kept in the presence of nocodazole to arrest them in mitosis. The presence of nocodazole did prevent the assembly of the three groups of genomes, the donor cell genome, the maternal and the paternal recipient cell genomes, into a single spindle.
  • zygotes transferred in interphase can reprogram an ES cell donor genome if their genome is removed in mitosis, but not if their nuclei are removed in interphase.
  • reprogramming activities are removed with the interphase nucleus but not with condensed mitotic chromosomes.
  • Example 16 Specific removal of chromatin instead of nuclei in interphase improves developmental potential of interphase cytoplasts
  • cytoplasts generated by specific removal of interphase chromatin are suitable recipients for an 8-cell stage nucleus, able to support development to the blastocyst stage and even to term after embryo transfer (Greda et al., Mouse zygotes as recipients in embryo cloning. Reproduction 132: 741-748, 2006). Whether such cytoplasts are able to reprogram cells of later stages of development has however not been addressed.
  • cytoplasts generated by specific enucleation is higher than that of cytoplasts generated by complete enucleation of the interphase nuclei (Fig. 13, Table 8).
  • cytoplasts generated in mitosis they failed to reprogram the genome of an embryonic stem cell (Fig. 13, Table 8).
  • Example 17 The transcriptional regulator Brg-1 is associated with chromatin in interphase but not in mitotic zygotes
  • Example 18 Developmental potential can be rescued by the transfer of nuclear factors
  • RNAs In contrast, nuclear factors such as Brg-1 are not removed with condensed chromatin in mitosis. If the presence of Brg-1 or other nuclear factors in the recipient cytoplast correlates with developmental potential, the transfer of nuclei but not mitotic chromosomes should confer a higher developmental potential to a recipient cytoplast devoid of these factors.
  • nuclei or mitotic genomes of zygotes were transferred into a zygote enucleated in interphase. Even though ovulation was stimulated with an hCG pulse at a single time point, a batch of zygotes does not enter mitosis synchronously. This allowed the transfer from M-phase to interphase zygotes between 28 and 33h post hCG.
  • Interphase zygotes with an M-phase genome entered mitosis within hours, cleaved to the 2-cell stage and thereafter invariably arrested (Table 8, Fig. 13). These zygotes formed a small nucleus without the prominent nucleoli usually contained in the nuclei of a 2-cell stage embryo (Fig. 16b). The transfer of zygote nuclei however was able to complement the missing factors and development was efficient (Fig. 13, Table 8). Nuclei or mitotic genomes were isolated from 8-cell stage blastomeres and then injected into zygotes enucleated in interphase.
  • Nuclear factors such as Brg-1 transferred with the 8-cell stage nucleus but not with the 8-cell stage mitotic genome may therefore partially complement the factors lost by enucleation of the zygotic interphase nucleus.
  • Entire mitotic ES cells or interphase ES cells, both of which do contain all factors required for cell cycle progression of an ES cell, were then injected into zygotes enucleated in interphase. These clones invariably arrested at the 1-cell or the 2-cell stage (Fig.13, Table 8). Factors contained in the ES cell therefore cannot complement the factors lost with the removal of the zygotic interphase nucleus.
  • Table 9 provides a summary of the developmental potentials of cloned zygotes constructed according to certain embodiments described herein. Table 6. Cell cycle synchronization by donor cell chromosome condensation is beneficial for the development of clones. morulae & recipient donor recipient
  • I interphase
  • M mitosis
  • A Anaphase of mitosis
  • zyg zygote
  • ES embryonic stem cell. shows the number of clones with interphase nuclei in the first interphase after transfer and inhibition of cytolcinesis.
  • Gl interphase synchronized by release from mitotic nocodazole arrest for Ih.
  • N/A not applicable.
  • P/D purvalanol A and 6-DMAP
  • Zygotes in interphase are suitable genome recipients if the host genome is removed in mitosis, or retained.
  • Table 9 A summary of the developmental potential of cloned zygotes constructed with somatic or ES cell genomes according to certain embodiments described herein
  • J Development occurs with donor nuclei of zygotes themselves or of a 2-cell stage embryo.
  • Example 19 Developmental reprogramming after transfer into mitotic blastomeres.
  • Blastomeres were arrested in mitosis with 0.1 ug/ml nocodazole. The mitotic genome was then removed in the presence of cytocholasinB and replaced with the genome of a donor cell as described for zygotes in the Examples above (see, e.g., Example 1). The genome of the donor cell was marked with H2B-cherry to follow it after transfer. The blastomeres then cleaved to two cells, which had red interphase nuclei. These cells contributed to the developing embryo to form bastocysts (Fig. 18). Stem cells could be derived from blastocysts generated in this manner (Fig. 19 a, b, c). These stem cells showed a normal karyotype and gave rise to high-percentage chimeric mice (Fig. 19d).
  • Somatic donor T cells do not show expression of an Oct-4 GFP transgene (Fig. 20a).
  • Fig. 20b Somatic donor T cells do not show expression of an Oct-4 GFP transgene.
  • Example 20 Somatic cell nuclear transfer into human zygotes
  • mouse zygotes can be used to reprogram the nucleus of a somatic cell.
  • FIG. 21 shows a human zygote in interphase.
  • Figure 21b, left panel shows a human zygote in protometaphase in the presence of 50 ug/ml nocodazole, added prior to nuclear envelope breakdown. The paternal and maternal genome are in two different locations.
  • Figure 21b, right panel shows removal of zygotic DNA without spindle formation.
  • Figure 21c shows staining for beta-tubulin, after removal of the genome without associated spindle material.
  • Figure 21d shows fusion of a somatic cell arrested in GO of the cell cycle to the zygote 2kV/cm DC pulse in electro fusion buffer.
  • Figure 21e shows condensation of somatic chromatin after transfer.

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Abstract

Selon certains modes de réalisation, l'invention concerne des procédés et des compositions utiles à la génération d'une cellule transgénique. Lesdits procédés comprennent le transfert de matériau génétique dérivé du noyau d'une cellule donneuse à un zygote fertilisé ou à un blastomère dont le matériau génétique dérivé du noyau a été retiré. L'invention concerne également des procédés et des compositions pour la génération de cellules souches embryonnaires transgéniques pluripotentes et d'animaux transgéniques, ainsi que des procédés d'utilisation de telles cellules souches embryonnaires transgéniques et d'animaux transgéniques pour une modélisation de maladie, un criblage de médicaments et/ou une thérapie par remplacement cellulaire.
PCT/US2008/061591 2007-04-26 2008-04-25 Dérivation de cellules souches embryonnaires WO2008134522A1 (fr)

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US20070033665A1 (en) * 1995-08-31 2007-02-08 Roslin (Edinburgh) Unactivated oocytes as cytoplast recipients for nuclear transfer

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WO2020204851A1 (fr) * 2019-04-05 2020-10-08 Celltek Sağlik Ve Danişmanlik Hi̇zmetleri̇ Li̇mi̇ted Şi̇rketi̇ Procédé de production de cellules souches induites

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