US20130217117A1 - Pluripotent stem cells obtained by non-viral reprogramming - Google Patents
Pluripotent stem cells obtained by non-viral reprogramming Download PDFInfo
- Publication number
- US20130217117A1 US20130217117A1 US13/607,072 US201213607072A US2013217117A1 US 20130217117 A1 US20130217117 A1 US 20130217117A1 US 201213607072 A US201213607072 A US 201213607072A US 2013217117 A1 US2013217117 A1 US 2013217117A1
- Authority
- US
- United States
- Prior art keywords
- pep4
- pcep4
- promoter
- cells
- ires2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/602—Sox-2
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/604—Klf-4
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/605—Nanog
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/606—Transcription factors c-Myc
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/608—Lin28
-
- 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
- C12N2510/00—Genetically modified cells
Definitions
- Embryonic stem (ES) cells hold great promise in science and medicine due to their pluripotent nature, i.e. the ability to replicate indefinitely and differentiate into cells of all three germ layers (Thomson et al., Science 282:1145-1147 (1998), incorporated by reference herein as if set forth in its entirety).
- the application of human ES cells in therapy and regenerative medicine is complicated by the possibility of rejection by the recipient's immune system.
- Human pluripotent cells that are substantially genetically identical to a particular recipient are, thus, highly desirable. Also, genetic identity may be important for the use of ES cells in designing patient-specific treatment strategies.
- iPS induced pluripotent
- iPS cells have been generated from a number of different human and murine somatic cell types, such as epithelial, fibroblast, liver, stomach, neural, and pancreatic cells. Further, iPS cells have been successfully differentiated into cells of various lineages (e.g., Dimos et al., Science 321:1218-1221 (2008)).
- Retroviral vectors such as those derived from lentivirus. These vectors stably integrate into, and permanently change, a target cell's DNA at virtually any chromosomal locus. This untargeted interaction between reprogramming vector and genome is associated with a risk of aberrant cellular gene expression as well as neoplastic growth caused by viral gene reactivation (Okita et al. Nature 448:313-317 (2007)).
- transgenes can interfere with the recipient cell's physiology.
- ectopic expression of transcription factors used to reprogram somatic cells can induce programmed cell death (apoptosis) (Askew et al., Oncogene 6:1915-1922 (1991), Evan et al., Cell 69:119-128 (1992)).
- continued expression of factors such as OCT4 can interfere with subsequent differentiation of iPS cells.
- the present invention is broadly summarized as relating to reprogramming of differentiated primate somatic cells to produce primate pluripotent cells.
- a method for producing a primate pluripotent cell includes the step of delivering into a primate somatic cell a set of transgenes sufficient to reprogram the somatic cell to a pluripotent state, the transgenes being carried on at least one episomal vector that does not encode an infectious virus, and recovering pluripotent cells.
- references herein to a “non-viral” vector or construct indicate that the vector or construct cannot encode an infectious virus.
- the invention in a second aspect, relates to an enriched population of replenishable reprogrammed pluripotent cells of a primate, including a human primate, wherein, in contrast to existing iPS cells, the at least one vector, including any element thereof having a viral source or derivation is substantially absent from the pluripotent cells.
- the reprogrammed cells contain fewer than one copy of the episomal vector per cell, and preferably no residual episomal vector in the cells. Because asymmetric partitioning during cell division dilutes the vector, one can readily obtain reprogrammed cells from which the vector has been lost.
- the primate pluripotent cells of the invention are substantially genetically identical to somatic cells from a fetal or post-natal individual. Fetal cells can be obtained from, e.g., amniotic fluid.
- the cells of the enriched population are not readily distinguished from existing primate ES and iPS cells morphologically (i.e., round shape, large nucleoli and scant cytoplasm) or by growth properties (i.e., doubling time; ES cells have a doubling time of about seventeen to eighteen hours).
- the reprogrammed cells Like iPS cells and ES cells, the reprogrammed cells also express pluripotent cell-specific markers (e.g., OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1). Unlike ES cells, the reprogrammed cells are not immediately derived from embryos. As used herein, “not immediately derived from embryos” means that the starting cell type for producing the pluripotent cells is a non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a fetal or post-natal individual. Like iPS cells, the pluripotent cells produced in the method can transiently express one or more copies of selected potency-determining factors during their derivation.
- pluripotent cell-specific markers e.g., OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1).
- FIG. 1A-B illustrate the effect on reprogramming efficiency of different nucleotide sequences that link transgenes on the vector(s) delivered during the reprogramming methods.
- FIG. 2A-C illustrate the effect on reprogramming efficiency of c-Myc, KLF-4, and SV40 large T antigen gene expression in human newborn foreskin fibroblasts.
- FIG. 3A-C illustrate a suitable construct for carrying transgenes into somatic cells in accord with the method, temporal expression of an episomal vector-mediated transgene, and the effect of vector quantity on cell survival after nucleofection.
- FIG. 4A-D illustrate reprogramming of human newborn foreskin fibroblasts with episomal vector-mediated transgene expression.
- FIG. 5A-B illustrate related constructs harboring an expression cassette useful in the reprogramming methods of the invention.
- the present invention broadly relates to novel methods for reprogramming differentiated primate somatic cells into reprogrammed primate cells that are substantially free of the vectors used in their production by introducing potency-determining factors on a non-viral vector that is present during reprogramming, but is substantially absent from the reprogrammed cells.
- reprogramming refers to a genetic process whereby differentiated somatic cells are converted into de-differentiated cells having a higher potency than the cells from which they were derived.
- the higher potency cells produced in the method are euploid pluripotent cells.
- pluripotent cells refer to a population of cells that express pluripotent cell-specific markers, have a cell morphology characteristic of undifferentiated cells (i.e., compact colony, high nucleus to cytoplasm ratio and prominent nucleolus) and can differentiate into all three germ layers (e.g., endoderm, mesoderm and ectoderm).
- the pluripotent cells form teratomas that typically contain cells or tissues characteristic of all three germ layers.
- One of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art.
- Pluripotent cells are capable of both proliferation in cell culture and differentiation towards a variety of lineage-restricted cell populations that exhibit multipotent properties. Pluripotent cells have a higher potency than somatic multipotent cells, which by comparison are more differentiated, but which are not terminally differentiated.
- the pluripotent products of primate somatic cell reprogramming methods are referred to herein as “reprogrammed primate pluripotent cells” or as induced pluripotent (iPS) cells.
- reprogrammed primate pluripotent cells or as induced pluripotent (iPS) cells.
- iPS induced pluripotent
- Differentiated somatic cells including cells from a fetal, newborn, juvenile or adult primate, including human, individual, are suitable starting cells in the methods.
- Suitable somatic cells include, but are not limited to, bone marrow cells, epithelial cells, endothelial cells, fibroblast cells, hematopoietic cells, keratinocytes, hepatic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells.
- Another suitable somatic cell is a CD29 + CD44 + CD166 + CD105 + CD73 + and CD31 ⁇ mesenchymal cell that attaches to a substrate.
- the somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells, muscle/bone stem cells, brain stem cells and liver stem cells.
- Suitable somatic cells are receptive, or can be made receptive using methods generally known in the scientific literature, to uptake of potency-determining factors including genetic material encoding the factors. Uptake-enhancing methods can vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive somatic cells having suitable transduction efficiency are well-known by those of ordinary skill in the art.
- the starting somatic cells can have a doubling time of about twenty-four hours.
- the vectors described herein can be constructed and engineered using methods generally known in the scientific literature to increase their safety for use in therapy, to include selection and enrichment markers, if desired, and to optimize expression of nucleotide sequences contained thereon.
- the vectors should include structural components that permit the vector to self-replicate in the somatic starting cells.
- Epstein Barr oriP/Nuclear Antigen-1 (EBNA-1) combination see, e.g., Lindner, S. E. and B.
- Plasmid 58:1 (2007), incorporated by reference as if set forth herein in its entirety is sufficient to support vector self-replication and other combinations known to function in mammalian, particularly primate, cells can also be employed.
- Standard techniques for the construction of expression vectors suitable for use in the present invention are well-known to one of ordinary skill in the art and can be found in publications such as Sambrook J, et al., “Molecular cloning: a laboratory manual,” (3rd ed. Cold Spring harbor Press, Cold Spring Harbor, N.Y. 2001), incorporated herein by reference as if set forth in its entirety.
- Suitable potency-determining factors can include, but are not limited to OCT-4, SOX2, LIN28, NANOG, c-Myc, KLF4, and combinations thereof.
- Each potency-determining factor can be introduced into the somatic cells as a polynucleotide transgene that encodes the potency-determining factor operably linked to a heterologous promoter that can drive expression of the polynucleotide in the somatic cell.
- SV40 T Antigen is not a potency-determining factor per se, it advantageously introduced into somatic cells as it provides the cells with a condition sufficient to promote cell survival during reprogramming while the potency-determining factors are expressed.
- Other conditions sufficient for expression of the factors include cell culture conditions described in the examples.
- Suitable reprogramming vectors are episomal vectors, such as plasmids, that do not encode all or part of a viral genome sufficient to give rise to an infectious or replication-competent virus, although the vectors can contain structural elements obtained from one or more virus.
- One or a plurality of reprogramming vectors can be introduced into a single somatic cell.
- One or more transgenes can be provided on a single reprogramming vector.
- One strong, constitutive transcriptional promoter can provide transcriptional control for a plurality of transgenes, which can be provided as an expression cassette.
- Separate expression cassettes on a vector can be under the transcriptional control of separate strong, constitutive promoters, which can be copies of the same promoter or can be distinct promoters.
- heterologous promoters are known in the art and can be used depending on factors such as the desired expression level of the potency-determining factor. It can be advantageous, as exemplified below, to control transcription of separate expression cassettes using distinct promoters having distinct strengths in the target somatic cells. Another consideration in selection of the transcriptional promoter(s) is the rate at which the promoter(s) is silenced in the target somatic cells. The skilled artisan will appreciate that it can be advantageous to reduce expression of one or more transgenes or transgene expression cassettes after the product of the gene(s) has completed or substantially completed its role in the reprogramming method.
- Exemplary promoters are the human EF1 ⁇ elongation factor promoter, CMV cytomegalovirus immediate early promoter and CAG chicken albumin promoter, and corresponding homologous promoters from other species.
- both EF1 ⁇ and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1 ⁇ promoter such that expression of transgenes under control of the former is turned off sooner than that of transgenes under control of the latter.
- the potency-determining factors can be expressed in the somatic cells in a relative ratio that can be varied to modulate reprogramming efficiency.
- somatic cell reprogramming efficiency is fourfold higher when OCT-4 and SOX2 are encoded in a single transcript on a single vector in a 1:1 ratio than when the two factors are provided on separate vectors, such that the uptake ratio of the factors into single cells is uncontrolled.
- an internal ribosome entry site is provided upstream of transgene(s) distal from the transcriptional promoter.
- the vectors can persist in target cells while the introduced transgenes are transcribed and translated. Transgene expression can be advantageously downregulated or turned off in cells that have been reprogrammed to a pluripotent state.
- the reprogramming vector(s) can remain extra-chromosomal. At extremely low efficiency, the vector(s) can integrate into the cells' genome.
- the reprogramming vector(s) replicate coordinately with the recipient cell's genome and, as such, are reasonably stable for about two weeks, longer than episomal vectors that cannot replicate their DNA.
- Pluripotent cells produced in the method can be cultured in any medium that supports pluripotent cell growth, including but not limited to a defined medium, such as TeSRTM (StemCell Technologies, Inc.; Vancouver, Canada), mTeSRTM (StemCell Technologies, Inc.) and StemLine® serum-free medium (Sigma; St. Louis, Mo.), or a conditioned medium such as mouse embryonic fibroblast (MEF)-conditioned medium.
- a “defined medium” refers to a biochemically defined formulation comprised solely of biochemically-defined constituents which can include constituents of known chemical composition or constituents derived from known sources.
- conditioned medium refers to a growth medium that is further supplemented with soluble factors from cells cultured in the medium. Alternatively, cells can be maintained on MEFs in culture medium.
- Suitable expression cassettes structures were created using conventional methods by direct polymerase chain reaction (PCR) amplification of open reading frames (ORFs) from some or all of the transgenes, using the first and last 20-22 bases of the coding region as primers, and from the Internal Ribosome Entry Sites listed in Table 1.
- ORFs open reading frames
- the sources of SV40 T Antigen and human telomerase reverse transcriptase, plasmids pBABE-puro SV40 LT and pBABE-hygro-hTERT, are commercially available from Addgene, Inc, Cambridge, Mass., as plasmids 13970 and 1773, respectively.
- IRES1 and IRES2 are commercially available from Clontech Laboratories, Inc., Mountain View, Calif. Foot-and-mouth disease virus segment 2, was chemically synthesized.
- In-frame expression cassettes are described using the codes set forth below in Table 1.
- E-O25 refers to an expression cassette having an EF1 ⁇ promoter upstream of the OCT4 and SOX2 coding regions, with IRES2 therebetween.
- C-M2K refers to an expression cassette having a CMV promoter upstream of the c-Myc and Klf4 coding regions, with IRES2 therebetween.
- O2S(2) a variant O2S expression cassette
- O2S(2) a variant O2S expression cassette
- TK promoter-Hyg-TK polyA cassette a variant O2S expression cassette
- FIGS. 5A and 5B Cassettes having the indicated structures were selected for subsequent use in reprogramming methods by empirical determination of expression levels of various factors.
- the promoter designated as EF2 (SEQ ID NO:12) was a slight variant from the known EF1 ⁇ promoter (SEQ ID NO:11) that did not differ from EF1 ⁇ in activity and which was not used in subsequent episomal vector reprogramming trials, infra.
- the F2A is a peptide linker that facilitates co-translation of distinct coding regions expressed from a single transcript. F2A was tested but was not used in subsequent reprogramming trials using episomal vectors. IRES 1 was tested but was not used in subsequent reprogramming trials using episomal vectors.
- FIG. 1A shows a Western blot analysis of OCT-4 and SOX2 in 293FT cells.
- Lane 1 pSIN4-EF2-OCT4-IRES1-SOX2; lane 2, pSIN4-EF2-OCT4-IRES2-SOX2; lane 3, pSIN4-EF2-OCT4-F2A-SOX2; lane 4, pSIN4-EF2-OCT4-IRES1-PURO; lane 5, pSIN4-EF2-SOX2-IRES1-PURO; lane 6, no plasmid (control).
- Mouse anti-human OCT4 monoclonal antibody (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., sc-5279) and goat anti-human SOX2 polyclonal antibody (1:500, R&D Systems, Minneapolis, Minn. AF2018) were used to detect the relative expression of OCT4 and SOX2 respectively.
- FIG. 1B shows reprogramming using linked potency-determining factors in 0.2 ⁇ 10 6 mesenchymal cells derived (Yu et al., supra) from OCT4 knock-in human ES cells (US Patent Application No. 2006/0128018 and Zwaka and Thomson, Nature Biotechnology 21:319-321 (2003), each incorporated herein by reference as if set forth in its entirety). This line was maintained under neomycin selection (geneticin: 100 ⁇ g/ml, Invitrogen Corp.). Human iPS cell colonies were counted on day 16 post-transduction.
- the gene combinations were pSIN4-EF2-OCT4-IRES1-SOX2 (O1S); pSIN4-EF2-OCT4-IRES2-SOX2 (O2S); pSIN4-EF2-OCT4-F2A-SOX2 (OF2AS); pSIN4-EF2-NANOG-IRES1-LIN28 (N1L); pSIN4-EF2-NANOG-IRES2-LIN28 (N2L); pSIN4-EF2-OCT4-IRES1-PURO (O); pSIN4-EF2-SOX2-IRES1-PURO (S); pSIN4-EF2-NANOG-IRESI-PURO (N); pSIN4-EF2-LIN28-IRES1-PURO (L).
- the abbreviation used for each lentiviral plasmid vector is shown in parentheses after the vector name.
- FIG. 2A shows that NANOG has a profound positive effect on reprogramming efficiency when OCT4, SOX2, LIN28, and c-MYC are also introduced, and that in combination with OCT4, SOX2, and LIN28, NANOG can support reprogramming, even in the absence of c-MYC or KLF4.
- Lentiviral constructs used were pSIN4-EF2-OCT4-IRES2-SOX2 (O2S); pSIN4-EF2-NANOG-IRES2-LIN28 (N2L); pSIN4-EF2-LIN28-IRES1-PURO (L); pSIN4-CMV-c-Myc-IRES1-PURO (M); pSIN4-EF2-KLF4-IRES1-PURO (K).
- FIG. 2B evidences reprogramming using linked potency-determining factors.
- Lentiviral constructs used were pSIN4-EF2-c-Myc-IRES2-KLF4 (EF2-M2K); pSIN4-CMV-c-Myc-IRES2-KLF4 (CMV-M2K); pSIN4-EF2-KLF4-IRES2-c-Myc (EF2-K2M); pSIN4-CMV-KLF4-IRES2-c-Myc (CMV-K2M); pSIN4-CMV-c-Myc-IRES2-LIN28 (M2L); pSIN4-EF2-NANOG-IRES2-KLF4 (N2K).
- alkaline phosphatase-positive human iPS cell colonies were counted. The number of iPS cell colonies were derived from an input of approximately 7.0 ⁇ 10 4 foreskin fibroblasts (passage 12). The asterisk indicates that most of the alkaline phosphatase-positive colonies appeared morphologically loose.
- FIG. 2C shows the effect of SV40 large T antigen gene expression on reprogramming efficiency.
- SV40 large T antigen prevents c-Myc-induced in murine fibroblasts (Hermeking et al., PNAS 91:10412-10416 (1994)) and enhances reprogramming efficiency (Hanna et al., Cell 133:250-264 (2008); Mali et al., Stem Cells doi: 10.1634/stemcells.2008-0346 (2008)).
- Abbreviations of gene combinations are the same as in FIG. 2B , with the addition of SV40 large T antigen (T).
- T SV40 large T antigen
- FIG. 2C demonstrates that if present at levels achieved during lentiviral-based reprogramming, T antigen inhibits final stages of iPS cell derivation. In contrast, see infra, wherein T antigen does not have this effect when present for the temporal expression time and/or level achieved during reprogramming using episomal vectors. In addition, T antigen prevents c-Myc-induced apoptosis but does not adversely affect c-Myc-induced cell proliferation.
- pCEP4-EGFP was created from commercially available mammalian episomal expression vector pCEP4 (Invitrogen Corp., Carlsbad, Calif.) by inserting the EGFP coding region between the pCEP4 BamHI and NheI sites.
- the episomal vectors of Table 2 were created by inserting the designated expression cassettes into pCEP4-EGFP or into a related backbone lacking P CMV (designated pEP4). See FIG. 3A and Table 2 footnotes for cloning sites into which expression cassettes were inserted.
- Vectors were introduced into the fibroblasts via a single nucleofection event, using Human Dermal Fibroblasts Nucleofector Kit (Normal Human Dermal Fibroblasts, Amaxa, Inc. Cat. No. VPD-1001), in accord with the manufacturer's instructions. After nucleofection, the transfected fibroblasts ( ⁇ 0.8 to 1.0 ⁇ 10 6 cells each) were immediately plated onto three 10 cm dishes seeded with irradiated mouse embryonic fibroblasts (MEF). Foreskin fibroblast culture medium was replaced every other day.
- Human Dermal Fibroblasts Nucleofector Kit Normal Human Dermal Fibroblasts, Amaxa, Inc. Cat. No. VPD-1001
- the foreskin fibroblast culture medium was replaced with human ES cell culture medium (DMEM/F12 culture medium supplemented with 20% KnockOut serum replacer, 0.1 mM non-essential amino acids (all from Invitrogen Corp.), 1 mM Glutamax, 0.1 mM ⁇ -mercaptoethanol and 100 ng/ml zebrafish basic fibroblast growth factor (zbFGF) as previously described (Amit et al., Developmental Biology 227:271-278 (2006); Ludwig et al., Nature Methods 3:637-646 (2006), each of which is incorporated herein by reference as if set forth in its entirety).
- human ES cell culture medium DMEM/F12 culture medium supplemented with 20% KnockOut serum replacer, 0.1 mM non-essential amino acids (all from Invitrogen Corp.), 1 mM Glutamax, 0.1 mM ⁇ -mercaptoethanol and 100 ng/ml zebrafish basic fibroblast growth factor (zbFGF
- human ES cell culture medium conditioned with irradiated MEF was used instead.
- the cultures were stained for alkaline phosphatase as an indication of human iPS colony development.
- temporal expression was initially evaluated by measuring EGFP level over time after introduction of EGFP from pEGFP-N2 (control) and pCEP4-EGFP episomal vector into 293FT cells was evaluated ( FIG. 3B ).
- FIG. 3C shows the effect of amount of pCEP4-EGFP episomal vector used on nucleofection efficiency and survival of human newborn foreskin fibroblasts, estimated from cell confluence on the day after nucleofection. Approximately 1 ⁇ 10 6 nucleofected foreskin fibroblasts were plated into each well of a 6-well plate. Gray lines represent non-transfected control fibroblasts; black lines represent transfected fibroblasts.
- FIG. 4A depicts schematic transgene expression constructs from Table 3 containing various expression cassettes that when introduced in certain combinations into human newborn foreskin fibroblasts result in reprogramming of the fibroblasts to pluripotent cells.
- Three combinations of introduced episomal reprogramming vectors have yielded reprogrammed pluripotent cells: (1) pEP4-E-O2S-E-T2K, pEP4-E-O2S-E-N2K and pCEP4-C-M2L; (2) pEP4-E-O2S-C-K2M-E-N2L and pEP4-E-O2S-E-T2K; and (3) pEP4-E-O2S-E-N2L, pEP4-E-O2S-E-T2K and pEP4-E-O2S-E-M2K.
- Table 3 indicates the amount of each vector used in each successful combination.
- FIG. 4B shows a bright-field microscopy image of a typical colony with morphological changes observed 18 days after episomal vector transfection.
- FIG. 4C shows a bright-field microscopy image of an alkaline phosphatase-positive colony 18 days after episomal vector transfection.
- FIG. 4D shows a bright-field microscopy image of a human iPS cell colony 6 days after the first passage of day 28 post-transfection reprogramming culture.
- the scale bar represents 0.1 mm.
- Reprogrammed cells were maintained for subsequent analysis in feeder-free culture on Matrigel (BD Biosciences, Bedford, Mass.) with conditioned medium as previously described (Xu et al., Nat. Biotechnol. 19:971 (2001), incorporated herein by reference as if set forth in its entirety).
- the reprogramming efficiency of greater than 1% of the newborn foreskin fibroblast cells reprogrammed was achieved, at significantly lower reprogramming time than was achieved using four gene combinations.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Transplantation (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Methods for reprogramming primate somatic cells to pluripotency using an episomal vector that does not encode an infectious virus are disclosed. Pluripotent cells produced in the methods are also disclosed.
Description
- This application is a continuation of U.S. patent application Ser. No. 12/605,220 filed Oct. 23, 2009, which will issue as U.S. Pat. No. 8,268,620 on Sep. 18, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61/108,362, filed Oct. 24, 2008, incorporated herein by reference as if set forth in its entirety.
- This invention was made with United States government support awarded by the following agencies: NIH GM081629, RR000167. The United States government has certain rights in this invention.
- Embryonic stem (ES) cells hold great promise in science and medicine due to their pluripotent nature, i.e. the ability to replicate indefinitely and differentiate into cells of all three germ layers (Thomson et al., Science 282:1145-1147 (1998), incorporated by reference herein as if set forth in its entirety). The application of human ES cells in therapy and regenerative medicine is complicated by the possibility of rejection by the recipient's immune system. Human pluripotent cells that are substantially genetically identical to a particular recipient are, thus, highly desirable. Also, genetic identity may be important for the use of ES cells in designing patient-specific treatment strategies.
- First attempts to generate pluripotent cells from a post-natal primate individual employed somatic nuclear transfer (see, e.g., Byrne, J A et al., Nature 450:497-502 (2007)) and cell fusion (see, e.g., Yu, J et al., Stem Cells 24:168-176 (2006)). However, clinical use of somatic nuclear transfer is impractical due to its low efficiency, while cell fusion results in near tetraploid cells. In 2007, two groups of scientists reprogrammed somatic cells from a post-natal primate individual into pluripotent stem cells (Yu et al., Science 318:1917-1920 (2007) and Takahashi et al., Cell 131:861-872 (2007)), each incorporated by reference herein as if set forth in its entirety. Both groups delivered into, and expressed in, human somatic cells cDNA of four transcription factors using a viral vector system for expressing potency-determining transgenes. The transcription factors of Takahashi et al. were OCT4, SOX2, c-Myc, and KLF4, while Yu et al. employed OCT4, SOX2, NANOG, and LIN28. The expression of these sets of transcription factors induced human somatic cells to acquire ES cell-specific characteristics, including morphology, proliferation, and gene- and surface marker expression. Somatic cells reprogrammed in this manner are referred to as induced pluripotent (iPS) cells. The existence of iPS cells circumvents the need for blastocysts and reduces concerns associated with immune rejection.
- Shortly thereafter, Lowry et al. generated patient-specific iPS cell lines through ectopic expression of OCT4, SOX2, c-Myc, and KLF4 (Lowry et al., PNAS 105:2883-2888 (2008)) transgenes. More recently, iPS cells have been generated from a number of different human and murine somatic cell types, such as epithelial, fibroblast, liver, stomach, neural, and pancreatic cells. Further, iPS cells have been successfully differentiated into cells of various lineages (e.g., Dimos et al., Science 321:1218-1221 (2008)).
- Current methods for generating iPS cells employ retroviral vectors such as those derived from lentivirus. These vectors stably integrate into, and permanently change, a target cell's DNA at virtually any chromosomal locus. This untargeted interaction between reprogramming vector and genome is associated with a risk of aberrant cellular gene expression as well as neoplastic growth caused by viral gene reactivation (Okita et al. Nature 448:313-317 (2007)).
- Moreover, continued presence and expression of the transgenes can interfere with the recipient cell's physiology. Further, ectopic expression of transcription factors used to reprogram somatic cells, such as c-Myc, can induce programmed cell death (apoptosis) (Askew et al., Oncogene 6:1915-1922 (1991), Evan et al., Cell 69:119-128 (1992)). Furthermore, continued expression of factors such as OCT4 can interfere with subsequent differentiation of iPS cells.
- It is desirable to reprogram somatic cells to a state of higher potency without altering the cells' genetic makeup beyond the reprogramming-associated alterations. Recently, Stadtfeld et al. generated murine iPS cells using a nonintegrating adenovirus that transiently expressed OCT4, SOX2, KLF4, and c-Myc (Stadtfeld et al., Sciencexpress, Sep. 25, 2008). To date, primate iPS cells generated without using retroviral vectors have not been reported.
- The present invention is broadly summarized as relating to reprogramming of differentiated primate somatic cells to produce primate pluripotent cells.
- In a first aspect, the invention is summarized in that a method for producing a primate pluripotent cell includes the step of delivering into a primate somatic cell a set of transgenes sufficient to reprogram the somatic cell to a pluripotent state, the transgenes being carried on at least one episomal vector that does not encode an infectious virus, and recovering pluripotent cells. References herein to a “non-viral” vector or construct indicate that the vector or construct cannot encode an infectious virus.
- In a second aspect, the invention relates to an enriched population of replenishable reprogrammed pluripotent cells of a primate, including a human primate, wherein, in contrast to existing iPS cells, the at least one vector, including any element thereof having a viral source or derivation is substantially absent from the pluripotent cells. As used herein, this means that the reprogrammed cells contain fewer than one copy of the episomal vector per cell, and preferably no residual episomal vector in the cells. Because asymmetric partitioning during cell division dilutes the vector, one can readily obtain reprogrammed cells from which the vector has been lost. As noted elsewhere herein, on very rare occasions a reprogramming vector can integrate into the genome of the cell, but cells having an integrated vector can be avoided by screening for absence of the vector. Further, in contrast to existing ES cells, the primate pluripotent cells of the invention are substantially genetically identical to somatic cells from a fetal or post-natal individual. Fetal cells can be obtained from, e.g., amniotic fluid. The cells of the enriched population are not readily distinguished from existing primate ES and iPS cells morphologically (i.e., round shape, large nucleoli and scant cytoplasm) or by growth properties (i.e., doubling time; ES cells have a doubling time of about seventeen to eighteen hours). Like iPS cells and ES cells, the reprogrammed cells also express pluripotent cell-specific markers (e.g., OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1). Unlike ES cells, the reprogrammed cells are not immediately derived from embryos. As used herein, “not immediately derived from embryos” means that the starting cell type for producing the pluripotent cells is a non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a fetal or post-natal individual. Like iPS cells, the pluripotent cells produced in the method can transiently express one or more copies of selected potency-determining factors during their derivation.
- Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable materials and methods for the practice or testing of the present invention are described below, other materials and methods similar or equivalent to those described herein, which are well known in the art, can be used.
- Other objectives, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.
-
FIG. 1A-B illustrate the effect on reprogramming efficiency of different nucleotide sequences that link transgenes on the vector(s) delivered during the reprogramming methods. -
FIG. 2A-C illustrate the effect on reprogramming efficiency of c-Myc, KLF-4, and SV40 large T antigen gene expression in human newborn foreskin fibroblasts. -
FIG. 3A-C illustrate a suitable construct for carrying transgenes into somatic cells in accord with the method, temporal expression of an episomal vector-mediated transgene, and the effect of vector quantity on cell survival after nucleofection. -
FIG. 4A-D illustrate reprogramming of human newborn foreskin fibroblasts with episomal vector-mediated transgene expression. -
FIG. 5A-B illustrate related constructs harboring an expression cassette useful in the reprogramming methods of the invention. - The present invention broadly relates to novel methods for reprogramming differentiated primate somatic cells into reprogrammed primate cells that are substantially free of the vectors used in their production by introducing potency-determining factors on a non-viral vector that is present during reprogramming, but is substantially absent from the reprogrammed cells. As used herein, “reprogramming” refers to a genetic process whereby differentiated somatic cells are converted into de-differentiated cells having a higher potency than the cells from which they were derived.
- Advantageously, the higher potency cells produced in the method are euploid pluripotent cells. As used herein, “pluripotent cells” refer to a population of cells that express pluripotent cell-specific markers, have a cell morphology characteristic of undifferentiated cells (i.e., compact colony, high nucleus to cytoplasm ratio and prominent nucleolus) and can differentiate into all three germ layers (e.g., endoderm, mesoderm and ectoderm). When introduced into an immunocompromised animal, such as a SCID mouse, the pluripotent cells form teratomas that typically contain cells or tissues characteristic of all three germ layers. One of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art. See, e.g., Thomson et al., supra. Pluripotent cells are capable of both proliferation in cell culture and differentiation towards a variety of lineage-restricted cell populations that exhibit multipotent properties. Pluripotent cells have a higher potency than somatic multipotent cells, which by comparison are more differentiated, but which are not terminally differentiated. The pluripotent products of primate somatic cell reprogramming methods are referred to herein as “reprogrammed primate pluripotent cells” or as induced pluripotent (iPS) cells. Such cells are suitable for use in research and therapeutic applications currently envisioned for human ES cells or existing iPS cells.
- Differentiated somatic cells, including cells from a fetal, newborn, juvenile or adult primate, including human, individual, are suitable starting cells in the methods. Suitable somatic cells include, but are not limited to, bone marrow cells, epithelial cells, endothelial cells, fibroblast cells, hematopoietic cells, keratinocytes, hepatic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells. Another suitable somatic cell is a CD29+ CD44+ CD166+ CD105+ CD73+ and CD31− mesenchymal cell that attaches to a substrate. Alternatively, the somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells, muscle/bone stem cells, brain stem cells and liver stem cells. Suitable somatic cells are receptive, or can be made receptive using methods generally known in the scientific literature, to uptake of potency-determining factors including genetic material encoding the factors. Uptake-enhancing methods can vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive somatic cells having suitable transduction efficiency are well-known by those of ordinary skill in the art. The starting somatic cells can have a doubling time of about twenty-four hours.
- The vectors described herein can be constructed and engineered using methods generally known in the scientific literature to increase their safety for use in therapy, to include selection and enrichment markers, if desired, and to optimize expression of nucleotide sequences contained thereon. The vectors should include structural components that permit the vector to self-replicate in the somatic starting cells. For example, the known Epstein Barr oriP/Nuclear Antigen-1 (EBNA-1) combination (see, e.g., Lindner, S. E. and B. Sugden, The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells, Plasmid 58:1 (2007), incorporated by reference as if set forth herein in its entirety) is sufficient to support vector self-replication and other combinations known to function in mammalian, particularly primate, cells can also be employed. Standard techniques for the construction of expression vectors suitable for use in the present invention are well-known to one of ordinary skill in the art and can be found in publications such as Sambrook J, et al., “Molecular cloning: a laboratory manual,” (3rd ed. Cold Spring harbor Press, Cold Spring Harbor, N.Y. 2001), incorporated herein by reference as if set forth in its entirety.
- In the methods, genetic material encoding a set of potency-determining factors is delivered into the somatic cells via one or more reprogramming vectors. Suitable potency-determining factors can include, but are not limited to OCT-4, SOX2, LIN28, NANOG, c-Myc, KLF4, and combinations thereof. Each potency-determining factor can be introduced into the somatic cells as a polynucleotide transgene that encodes the potency-determining factor operably linked to a heterologous promoter that can drive expression of the polynucleotide in the somatic cell. Although SV40 T Antigen is not a potency-determining factor per se, it advantageously introduced into somatic cells as it provides the cells with a condition sufficient to promote cell survival during reprogramming while the potency-determining factors are expressed. Other conditions sufficient for expression of the factors include cell culture conditions described in the examples.
- Suitable reprogramming vectors are episomal vectors, such as plasmids, that do not encode all or part of a viral genome sufficient to give rise to an infectious or replication-competent virus, although the vectors can contain structural elements obtained from one or more virus. One or a plurality of reprogramming vectors can be introduced into a single somatic cell. One or more transgenes can be provided on a single reprogramming vector. One strong, constitutive transcriptional promoter can provide transcriptional control for a plurality of transgenes, which can be provided as an expression cassette. Separate expression cassettes on a vector can be under the transcriptional control of separate strong, constitutive promoters, which can be copies of the same promoter or can be distinct promoters. Various heterologous promoters are known in the art and can be used depending on factors such as the desired expression level of the potency-determining factor. It can be advantageous, as exemplified below, to control transcription of separate expression cassettes using distinct promoters having distinct strengths in the target somatic cells. Another consideration in selection of the transcriptional promoter(s) is the rate at which the promoter(s) is silenced in the target somatic cells. The skilled artisan will appreciate that it can be advantageous to reduce expression of one or more transgenes or transgene expression cassettes after the product of the gene(s) has completed or substantially completed its role in the reprogramming method. Exemplary promoters are the human EF1α elongation factor promoter, CMV cytomegalovirus immediate early promoter and CAG chicken albumin promoter, and corresponding homologous promoters from other species. In human somatic cells, both EF1α and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1α promoter such that expression of transgenes under control of the former is turned off sooner than that of transgenes under control of the latter.
- The potency-determining factors can be expressed in the somatic cells in a relative ratio that can be varied to modulate reprogramming efficiency. For example, somatic cell reprogramming efficiency is fourfold higher when OCT-4 and SOX2 are encoded in a single transcript on a single vector in a 1:1 ratio than when the two factors are provided on separate vectors, such that the uptake ratio of the factors into single cells is uncontrolled. Preferably, where a plurality of transgenes is encoded on a single transcript, an internal ribosome entry site is provided upstream of transgene(s) distal from the transcriptional promoter. Although the relative ratio of factors can vary depending upon the factors delivered, one of ordinary skill in possession of this disclosure can determine an optimal ratio of factors.
- The skilled artisan will appreciate that the advantageous efficiency of introducing all factors via a single vector rather than via a plurality of vectors, but that as total vector size increases, it becomes increasingly difficult to introduce the vector. The skilled artisan will also appreciate that position of a factor on a vector can affect its temporal expression, and the resulting reprogramming efficiency. As such, Applicants employed various combinations of factors on combinations of vectors. Several such combinations are here shown to support reprogramming.
- After introduction of the reprogramming vector(s) and while the somatic cells are being reprogrammed, the vectors can persist in target cells while the introduced transgenes are transcribed and translated. Transgene expression can be advantageously downregulated or turned off in cells that have been reprogrammed to a pluripotent state. The reprogramming vector(s) can remain extra-chromosomal. At extremely low efficiency, the vector(s) can integrate into the cells' genome. The reprogramming vector(s) replicate coordinately with the recipient cell's genome and, as such, are reasonably stable for about two weeks, longer than episomal vectors that cannot replicate their DNA. Nevertheless, because the vectors are not partitioned evenly at cell division, in the absence of selective pressure, cells lose the episomal vector(s) so one can readily recover vector-free pluripotent cells in the method. For example, it usually takes two-to-three weeks for oriP/EBNA-1-based episomal plasmids to be stably maintained in somatic cells. During the initial two-to-three weeks, cells quickly lose episomal plasmids. Once the cells are stabilized, the cells continue to lose episomal vector at ˜5% per generation.
- Pluripotent cells produced in the method can be cultured in any medium that supports pluripotent cell growth, including but not limited to a defined medium, such as TeSR™ (StemCell Technologies, Inc.; Vancouver, Canada), mTeSR™ (StemCell Technologies, Inc.) and StemLine® serum-free medium (Sigma; St. Louis, Mo.), or a conditioned medium such as mouse embryonic fibroblast (MEF)-conditioned medium. As used herein, a “defined medium” refers to a biochemically defined formulation comprised solely of biochemically-defined constituents which can include constituents of known chemical composition or constituents derived from known sources. As used herein, “conditioned medium” refers to a growth medium that is further supplemented with soluble factors from cells cultured in the medium. Alternatively, cells can be maintained on MEFs in culture medium.
- The invention will be more fully understood upon consideration of the following non-limiting Examples.
- Suitable expression cassettes structures were created using conventional methods by direct polymerase chain reaction (PCR) amplification of open reading frames (ORFs) from some or all of the transgenes, using the first and last 20-22 bases of the coding region as primers, and from the Internal Ribosome Entry Sites listed in Table 1. The sources of SV40 T Antigen and human telomerase reverse transcriptase, plasmids pBABE-puro SV40 LT and pBABE-hygro-hTERT, are commercially available from Addgene, Inc, Cambridge, Mass., as plasmids 13970 and 1773, respectively. The sources of IRES1 and IRES2, plasmids pIRESpuro3 and pIRES2EGFP, are commercially available from Clontech Laboratories, Inc., Mountain View, Calif. Foot-and-mouth
disease virus segment 2, was chemically synthesized. In-frame expression cassettes are described using the codes set forth below in Table 1. For example, “E-O25” refers to an expression cassette having an EF1α promoter upstream of the OCT4 and SOX2 coding regions, with IRES2 therebetween. Likewise, “C-M2K” refers to an expression cassette having a CMV promoter upstream of the c-Myc and Klf4 coding regions, with IRES2 therebetween. In several constructs, none of which was used in subsequent reprogramming, a variant O2S expression cassette (“O2S(2)”) was employed that differed from O2S in that it contained a TK promoter-Hyg-TK polyA cassette (compareFIGS. 5A and 5B ). Cassettes having the indicated structures were selected for subsequent use in reprogramming methods by empirical determination of expression levels of various factors. The promoter designated as EF2 (SEQ ID NO:12) was a slight variant from the known EF1α promoter (SEQ ID NO:11) that did not differ from EF1α in activity and which was not used in subsequent episomal vector reprogramming trials, infra. The F2A is a peptide linker that facilitates co-translation of distinct coding regions expressed from a single transcript. F2A was tested but was not used in subsequent reprogramming trials using episomal vectors.IRES 1 was tested but was not used in subsequent reprogramming trials using episomal vectors. - The relative effects of various promoters, IRES sequences, and transgene arrangements on the expression of the upstream and downstream ORFs were evaluated by separately cloning various transgene expression cassettes into pSin4, a modified lentivirus-based vector, to test their ability to reprogram human somatic cells after transfection, as previously described (Yu et al., supra). 293FT cells were transfected with lentiviral plasmid vectors expressing OCT4 and SOX2 linked by IRES1 or IRES2 using SuperFect (Qiagen, Valencia, Calif.), as depicted below. Cells were collected two days post-transfection.
FIG. 1A shows a Western blot analysis of OCT-4 and SOX2 in 293FT cells.Lane 1, pSIN4-EF2-OCT4-IRES1-SOX2;lane 2, pSIN4-EF2-OCT4-IRES2-SOX2;lane 3, pSIN4-EF2-OCT4-F2A-SOX2;lane 4, pSIN4-EF2-OCT4-IRES1-PURO;lane 5, pSIN4-EF2-SOX2-IRES1-PURO;lane 6, no plasmid (control). Mouse anti-human OCT4 monoclonal antibody (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., sc-5279) and goat anti-human SOX2 polyclonal antibody (1:500, R&D Systems, Minneapolis, Minn. AF2018) were used to detect the relative expression of OCT4 and SOX2 respectively. -
FIG. 1B shows reprogramming using linked potency-determining factors in 0.2×106 mesenchymal cells derived (Yu et al., supra) from OCT4 knock-in human ES cells (US Patent Application No. 2006/0128018 and Zwaka and Thomson, Nature Biotechnology 21:319-321 (2003), each incorporated herein by reference as if set forth in its entirety). This line was maintained under neomycin selection (geneticin: 100 μg/ml, Invitrogen Corp.). Human iPS cell colonies were counted on day 16 post-transduction. The gene combinations were pSIN4-EF2-OCT4-IRES1-SOX2 (O1S); pSIN4-EF2-OCT4-IRES2-SOX2 (O2S); pSIN4-EF2-OCT4-F2A-SOX2 (OF2AS); pSIN4-EF2-NANOG-IRES1-LIN28 (N1L); pSIN4-EF2-NANOG-IRES2-LIN28 (N2L); pSIN4-EF2-OCT4-IRES1-PURO (O); pSIN4-EF2-SOX2-IRES1-PURO (S); pSIN4-EF2-NANOG-IRESI-PURO (N); pSIN4-EF2-LIN28-IRES1-PURO (L). The abbreviation used for each lentiviral plasmid vector is shown in parentheses after the vector name. - Preliminary reprogramming experiments were conducted by introducing lentiviral vectors into human neonatal foreskin fibroblasts.
FIG. 2A shows that NANOG has a profound positive effect on reprogramming efficiency when OCT4, SOX2, LIN28, and c-MYC are also introduced, and that in combination with OCT4, SOX2, and LIN28, NANOG can support reprogramming, even in the absence of c-MYC or KLF4. Lentiviral constructs used were pSIN4-EF2-OCT4-IRES2-SOX2 (O2S); pSIN4-EF2-NANOG-IRES2-LIN28 (N2L); pSIN4-EF2-LIN28-IRES1-PURO (L); pSIN4-CMV-c-Myc-IRES1-PURO (M); pSIN4-EF2-KLF4-IRES1-PURO (K). Twenty-one days after transduction, alkaline phosphatase-positive human iPS cell colonies were counted. The number of iPS cell colonies were derived from an input of 2.5×104 human newborn foreskin fibroblasts (passage 9). The light gray bars represent the total number of reprogrammed colonies formed having typical human ES cell morphology; dark gray bars indicate the number of large colonies with minimal differentiation. -
FIG. 2B evidences reprogramming using linked potency-determining factors. Lentiviral constructs used were pSIN4-EF2-c-Myc-IRES2-KLF4 (EF2-M2K); pSIN4-CMV-c-Myc-IRES2-KLF4 (CMV-M2K); pSIN4-EF2-KLF4-IRES2-c-Myc (EF2-K2M); pSIN4-CMV-KLF4-IRES2-c-Myc (CMV-K2M); pSIN4-CMV-c-Myc-IRES2-LIN28 (M2L); pSIN4-EF2-NANOG-IRES2-KLF4 (N2K). Fourteen days after transduction, alkaline phosphatase-positive human iPS cell colonies were counted. The number of iPS cell colonies were derived from an input of approximately 7.0×104 foreskin fibroblasts (passage 12). The asterisk indicates that most of the alkaline phosphatase-positive colonies appeared morphologically loose. -
FIG. 2C shows the effect of SV40 large T antigen gene expression on reprogramming efficiency. SV40 large T antigen prevents c-Myc-induced in murine fibroblasts (Hermeking et al., PNAS 91:10412-10416 (1994)) and enhances reprogramming efficiency (Hanna et al., Cell 133:250-264 (2008); Mali et al., Stem Cells doi: 10.1634/stemcells.2008-0346 (2008)). Abbreviations of gene combinations are the same as inFIG. 2B , with the addition of SV40 large T antigen (T). c-Myc also promotes cell proliferation. Twelve days after transduction, alkaline phosphatase-positive human iPS cell colonies were counted. The number of iPS cell colonies were derived from an input of approximately ˜3.5×104 foreskin fibroblasts (passage 17).FIG. 2C demonstrates that if present at levels achieved during lentiviral-based reprogramming, T antigen inhibits final stages of iPS cell derivation. In contrast, see infra, wherein T antigen does not have this effect when present for the temporal expression time and/or level achieved during reprogramming using episomal vectors. In addition, T antigen prevents c-Myc-induced apoptosis but does not adversely affect c-Myc-induced cell proliferation. - Human newborn foreskin fibroblasts (Cat #CRL-2097™, ATCC) were maintained in foreskin fibroblast culture medium (DMEM (Cat #11965, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, Logan, Utah), 2 mM Glutamax, 0.1 mM non-essential amino acids, and 0.1 mM β-mercaptoethanol).
- Various combinations of potency-determining factors provided as transgene expression cassettes constructed as in Example 1 and as detailed below in Table 3 were introduced into somatic cells using an episomal construct pCEP4-EGFP (as shown in
FIG. 3A ) resulting in reprogramming with varying efficiency. pCEP4-EGFP was created from commercially available mammalian episomal expression vector pCEP4 (Invitrogen Corp., Carlsbad, Calif.) by inserting the EGFP coding region between the pCEP4 BamHI and NheI sites. The episomal vectors of Table 2 were created by inserting the designated expression cassettes into pCEP4-EGFP or into a related backbone lacking PCMV (designated pEP4). SeeFIG. 3A and Table 2 footnotes for cloning sites into which expression cassettes were inserted. - Vectors were introduced into the fibroblasts via a single nucleofection event, using Human Dermal Fibroblasts Nucleofector Kit (Normal Human Dermal Fibroblasts, Amaxa, Inc. Cat. No. VPD-1001), in accord with the manufacturer's instructions. After nucleofection, the transfected fibroblasts (˜0.8 to 1.0×106 cells each) were immediately plated onto three 10 cm dishes seeded with irradiated mouse embryonic fibroblasts (MEF). Foreskin fibroblast culture medium was replaced every other day. After four days, the foreskin fibroblast culture medium was replaced with human ES cell culture medium (DMEM/F12 culture medium supplemented with 20% KnockOut serum replacer, 0.1 mM non-essential amino acids (all from Invitrogen Corp.), 1 mM Glutamax, 0.1 mM β-mercaptoethanol and 100 ng/ml zebrafish basic fibroblast growth factor (zbFGF) as previously described (Amit et al., Developmental Biology 227:271-278 (2006); Ludwig et al., Nature Methods 3:637-646 (2006), each of which is incorporated herein by reference as if set forth in its entirety). When the seeded MEF could no longer sustain the reprogramming culture, about 8 to 10 days after plating, human ES cell culture medium conditioned with irradiated MEF was used instead. When appropriate (about 2-3 weeks after transfection), the cultures were stained for alkaline phosphatase as an indication of human iPS colony development.
- To determine suitable parameters for introducing transgene constructs, temporal expression was initially evaluated by measuring EGFP level over time after introduction of EGFP from pEGFP-N2 (control) and pCEP4-EGFP episomal vector into 293FT cells was evaluated (
FIG. 3B ). - The effect of the amount of transgene construct introduced on human newborn foreskin fibroblast cell survival was also evaluated in preliminary experiments.
FIG. 3C shows the effect of amount of pCEP4-EGFP episomal vector used on nucleofection efficiency and survival of human newborn foreskin fibroblasts, estimated from cell confluence on the day after nucleofection. Approximately 1×106 nucleofected foreskin fibroblasts were plated into each well of a 6-well plate. Gray lines represent non-transfected control fibroblasts; black lines represent transfected fibroblasts. -
FIG. 4A depicts schematic transgene expression constructs from Table 3 containing various expression cassettes that when introduced in certain combinations into human newborn foreskin fibroblasts result in reprogramming of the fibroblasts to pluripotent cells. Three combinations of introduced episomal reprogramming vectors have yielded reprogrammed pluripotent cells: (1) pEP4-E-O2S-E-T2K, pEP4-E-O2S-E-N2K and pCEP4-C-M2L; (2) pEP4-E-O2S-C-K2M-E-N2L and pEP4-E-O2S-E-T2K; and (3) pEP4-E-O2S-E-N2L, pEP4-E-O2S-E-T2K and pEP4-E-O2S-E-M2K. Table 3 indicates the amount of each vector used in each successful combination. One vector in each successful reprogramming combination encoded T antigen under control of the EF1α promoter. -
FIG. 4B shows a bright-field microscopy image of a typical colony with morphological changes observed 18 days after episomal vector transfection.FIG. 4C shows a bright-field microscopy image of an alkaline phosphatase-positive colony 18 days after episomal vector transfection. - Twenty-five to thirty days after transfection, the reprogramming cultures were passaged once to fresh 10 cm MEF dishes (1:3 ratio), due to the presence of many non-iPS cell colonies with morphologies similar to human iPS cell colonies. Colonies were then picked for further analysis.
FIG. 4D shows a bright-field microscopy image of a humaniPS cell colony 6 days after the first passage of day 28 post-transfection reprogramming culture. The scale bar represents 0.1 mm. Reprogrammed cells were maintained for subsequent analysis in feeder-free culture on Matrigel (BD Biosciences, Bedford, Mass.) with conditioned medium as previously described (Xu et al., Nat. Biotechnol. 19:971 (2001), incorporated herein by reference as if set forth in its entirety). - Advantageously, the reprogramming efficiency of greater than 1% of the newborn foreskin fibroblast cells reprogrammed was achieved, at significantly lower reprogramming time than was achieved using four gene combinations.
- It is understood that certain adaptations of the invention described in this disclosure are a matter of routine optimization for those skilled in the art, and can be implemented without departing from the spirit of the invention, or the scope of the appended claims. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. It is understood, however, that examples and embodiments of the present invention set forth above are illustrative and not intended to confine the invention. The invention embraces all modified forms of the examples and embodiments as come with the scope of the following claims.
Claims (20)
1. A population of pluripotent cells produced according to a method comprising the steps of:
introducing into a primate somatic cell at least one non-viral episomal vector that encodes at least one potency-determining factor under conditions sufficient to reprogram the cell into a pluripotent cell, the at least one vector encoding at least potency-determining factors OCT4 and SOX2, wherein the cell expresses the introduced OCT4 and SOX2 and further comprises SV40 T Antigen; and
culturing the pluripotent cell until it divides into a population of pluripotent cells substantially free of any vector component associated with exposing the potency-determining factors to the somatic cell.
2. The population of claim 1 , where the primate somatic cells are obtained from a fetal or post-natal primate individual.
3. The population of claim 2 , wherein the primate is human.
4. The population of claim 1 , wherein the pluripotent cells are genetically identical to a post-natal individual.
5. The population of claim 1 , wherein at least one vector further encodes at least one potency-determining factor selected from the group consisting of LIN28, NANOG, c-Myc, and KLF4.
6. The population of claim 1 , wherein a single vector encodes at least OCT4 and SOX2 and comprises at least one operably-positioned internal ribosome entry site.
7. The population of claim 6 , wherein the single vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4.
8. The population of claim 6 , wherein the vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, c-Myc, IRES2, KLF4, a third promoter, NANOG, IRES2, and LIN28.
9. The population of claim 1 , wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, NANOG, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, c-Myc, IRES 2, and LIN28, wherein the promoters need not be identical.
10. The population of claim 9 , wherein each of the first, second, third, and fourth promoters is an elongation factor 1α (EF1α) gene promoter.
11. The population of claim 1 , wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, KLF4, IRES2, c-Myc, a third promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a fourth promoter, OCT4, IRES2, SOX2, a fifth promoter, SV40 T antigen, IRES2, and KLF4, wherein the promoters need not be identical.
12. The population of claim 11 , wherein each of the first, third, fourth, and fifth promoters is an EF1α gene promoter and wherein the second promoter is a cytomegalovirus immediate early gene (CMV) promoter.
13. The population of claim 1 wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, SV40 T antigen, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, OCT4, IRES2, SOX2, a sixth promoter, c-Myc, IRES 2, and KLF4, wherein the promoters need not be identical.
14. The population of claim 1 , wherein at least one vector encodes a plurality of factors and at least one vector comprises at least one internal ribosomal entry site.
15. The population of claim 1 , wherein OCT4 and SOX2 are human.
16. The population of claim 1 , wherein LIN28, NANOG, c-Myc, and KLF4 are human.
17. A population of pluripotent cells substantially cured of exogenously introduced vector components.
18. The population of claim 17 , wherein the cells are primate cells.
19. The population of claim 18 , wherein the primate cells are human cells.
20. The population of claim 17 , wherein the human cells are genetically identical to cells of a post-natal primate individual.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/607,072 US20130217117A1 (en) | 2008-10-24 | 2012-09-07 | Pluripotent stem cells obtained by non-viral reprogramming |
US16/209,722 US20190330654A1 (en) | 2008-10-24 | 2018-12-04 | Pluripotent stem cells obtained by non-viral reprogramming |
US17/352,873 US20220010331A1 (en) | 2008-10-24 | 2021-06-21 | Pluripotent stem cells obtained by non-viral reporgramming |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10836208P | 2008-10-24 | 2008-10-24 | |
US12/605,220 US8268620B2 (en) | 2008-10-24 | 2009-10-23 | OCT4 and SOX2 with SV40 T antigen produce pluripotent stem cells from primate somatic cells |
US13/607,072 US20130217117A1 (en) | 2008-10-24 | 2012-09-07 | Pluripotent stem cells obtained by non-viral reprogramming |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/605,220 Continuation US8268620B2 (en) | 2008-10-24 | 2009-10-23 | OCT4 and SOX2 with SV40 T antigen produce pluripotent stem cells from primate somatic cells |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/209,722 Continuation US20190330654A1 (en) | 2008-10-24 | 2018-12-04 | Pluripotent stem cells obtained by non-viral reprogramming |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130217117A1 true US20130217117A1 (en) | 2013-08-22 |
Family
ID=41698343
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/605,220 Active 2029-12-10 US8268620B2 (en) | 2008-10-24 | 2009-10-23 | OCT4 and SOX2 with SV40 T antigen produce pluripotent stem cells from primate somatic cells |
US13/607,072 Abandoned US20130217117A1 (en) | 2008-10-24 | 2012-09-07 | Pluripotent stem cells obtained by non-viral reprogramming |
US16/209,722 Abandoned US20190330654A1 (en) | 2008-10-24 | 2018-12-04 | Pluripotent stem cells obtained by non-viral reprogramming |
US17/352,873 Pending US20220010331A1 (en) | 2008-10-24 | 2021-06-21 | Pluripotent stem cells obtained by non-viral reporgramming |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/605,220 Active 2029-12-10 US8268620B2 (en) | 2008-10-24 | 2009-10-23 | OCT4 and SOX2 with SV40 T antigen produce pluripotent stem cells from primate somatic cells |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/209,722 Abandoned US20190330654A1 (en) | 2008-10-24 | 2018-12-04 | Pluripotent stem cells obtained by non-viral reprogramming |
US17/352,873 Pending US20220010331A1 (en) | 2008-10-24 | 2021-06-21 | Pluripotent stem cells obtained by non-viral reporgramming |
Country Status (10)
Country | Link |
---|---|
US (4) | US8268620B2 (en) |
EP (2) | EP3450545B1 (en) |
JP (5) | JP2012506702A (en) |
CN (2) | CN102239249A (en) |
CA (1) | CA2741090C (en) |
DK (2) | DK3450545T3 (en) |
ES (1) | ES2959327T3 (en) |
IL (1) | IL212433B (en) |
SG (1) | SG10201600234PA (en) |
WO (1) | WO2010048567A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9175268B2 (en) | 2008-08-12 | 2015-11-03 | Cellular Dynamics International, Inc. | Methods for the production of iPS cells |
US9328332B2 (en) | 2008-06-04 | 2016-05-03 | Cellular Dynamics International, Inc. | Methods for the production of IPS cells using non-viral approach |
WO2016069282A1 (en) * | 2014-10-31 | 2016-05-06 | The Trustees Of The University Of Pennsylvania | Altering gene expression in modified t cells and uses thereof |
US9499786B2 (en) | 2007-03-23 | 2016-11-22 | Wisconsin Alumni Research Foundation | Enriched population of human pluripotent cells with Oct-4 and Sox2 integrated into their genome |
US10221395B2 (en) | 2016-06-16 | 2019-03-05 | Cedars-Sinai Medical Center | Efficient method for reprogramming blood to induced pluripotent stem cells |
WO2020051453A1 (en) | 2018-09-07 | 2020-03-12 | Wisconsin Alumni Research Foundation | Generation of hematopoietic progenitor cells from human pluripotent stem cells |
US10738323B2 (en) | 2013-07-12 | 2020-08-11 | Cedars-Sinai Medical Center | Generation of induced pluripotent stem cells from normal human mammary epithelial cells |
US10760057B2 (en) | 2017-07-06 | 2020-09-01 | Wisconsin Alumni Research Foundation | Human pluripotent stem cell-based screening for smooth muscle cell differentiation and disease |
US11464182B2 (en) | 2015-07-02 | 2022-10-11 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Method of inducing genetic recombination, and use therefor |
US11572545B2 (en) | 2016-06-16 | 2023-02-07 | Cedars-Sinai Medical Center | Efficient method for reprogramming blood to induced pluripotent stem cells |
Families Citing this family (68)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8129187B2 (en) | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
US20090227032A1 (en) * | 2005-12-13 | 2009-09-10 | Kyoto University | Nuclear reprogramming factor and induced pluripotent stem cells |
US8278104B2 (en) * | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
CN103113463B (en) | 2005-12-13 | 2015-02-18 | 国立大学法人京都大学 | Nuclear reprogramming factor |
JP2008307007A (en) | 2007-06-15 | 2008-12-25 | Bayer Schering Pharma Ag | Human pluripotent stem cell induced from human tissue-originated undifferentiated stem cell after birth |
US9213999B2 (en) * | 2007-06-15 | 2015-12-15 | Kyoto University | Providing iPSCs to a customer |
JP5346925B2 (en) | 2008-05-02 | 2013-11-20 | 国立大学法人京都大学 | Nuclear initialization method |
CN102239249A (en) * | 2008-10-24 | 2011-11-09 | 威斯康星校友研究基金会 | Pluripotent stem cells obtained by non-viral reprogramming |
AU2010254811B2 (en) | 2009-06-05 | 2015-02-19 | FUJIFILM Cellular Dynamics, Inc. | Reprogramming T cells and hematopoietic cells |
US8048675B1 (en) * | 2010-05-12 | 2011-11-01 | Ipierian, Inc. | Integration-free human induced pluripotent stem cells from blood |
EP2582794B2 (en) | 2010-06-15 | 2024-04-24 | FUJIFILM Cellular Dynamics, Inc. | Generation of induced pluripotent stem cells from small volumes of peripheral blood |
US9279103B2 (en) * | 2010-08-05 | 2016-03-08 | Wisconsin Alumni Research Foundation | Simplified basic media for human pluripotent cell culture |
US9279107B2 (en) | 2010-08-05 | 2016-03-08 | Wisconsin Alumni Research Foundation | Simplified basic media for human pluripotent cell culture |
JP2013545439A (en) | 2010-09-17 | 2013-12-26 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Functional genomics assay to characterize the usefulness and safety of pluripotent stem cells |
US9133266B2 (en) | 2011-05-06 | 2015-09-15 | Wisconsin Alumni Research Foundation | Vitronectin-derived cell culture substrate and uses thereof |
WO2013009825A1 (en) * | 2011-07-11 | 2013-01-17 | Cellular Dynamics International, Inc. | Methods for cell reprogramming and genome engineering |
JP6466170B2 (en) | 2011-10-17 | 2019-02-06 | ミネルバ バイオテクノロジーズ コーポレーション | Medium for stem cell growth and induction |
US10391126B2 (en) | 2011-11-18 | 2019-08-27 | Board Of Regents, The University Of Texas System | CAR+ T cells genetically modified to eliminate expression of T-cell receptor and/or HLA |
US8497124B2 (en) | 2011-12-05 | 2013-07-30 | Factor Bioscience Inc. | Methods and products for reprogramming cells to a less differentiated state |
EP3835420A1 (en) | 2011-12-05 | 2021-06-16 | Factor Bioscience Inc. | Methods and products for transfecting cells |
US8772460B2 (en) | 2011-12-16 | 2014-07-08 | Wisconsin Alumni Research Foundation | Thermostable FGF-2 mutant having enhanced stability |
US20130266541A1 (en) * | 2012-04-06 | 2013-10-10 | The Johns Hopkins University | Human induced pluripotent stem cells |
RU2711249C2 (en) | 2012-11-01 | 2020-01-15 | Фэктор Байосайенс Инк. | Methods and products for expression of proteins in cells |
DK2951290T3 (en) | 2013-02-01 | 2018-01-22 | Us Health | PROCEDURE FOR PREPARING RETINAL PIGMENTAL EPIT (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCS) |
EP3008229B1 (en) | 2013-06-10 | 2020-05-27 | President and Fellows of Harvard College | Early developmental genomic assay for characterizing pluripotent stem cell utility and safety |
KR101551926B1 (en) | 2013-09-06 | 2015-09-10 | 가톨릭대학교 산학협력단 | Human induced pluripotent stem cells and method for producing animal expressed human immune system using the same |
US11377639B2 (en) | 2013-11-15 | 2022-07-05 | Wisconsin Alumni Research Foundation | Lineage reprogramming to induced cardiac progenitor cells (iCPC) by defined factors |
EP4249036A3 (en) | 2014-01-31 | 2023-10-25 | Factor Bioscience Inc. | Methods and products for nucleic acid production and delivery |
KR20160145186A (en) * | 2014-04-24 | 2016-12-19 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Application of induced pluripotent stem cells to generate adoptive cell therapy products |
EP3543339A1 (en) | 2015-02-13 | 2019-09-25 | Factor Bioscience Inc. | Nucleic acid products and methods of administration thereof |
EP3296390B1 (en) * | 2015-04-14 | 2023-01-04 | Kyoto University | Method for producing stem cell clones suitable for induction of differentiation into somatic cells |
AU2016321170B2 (en) | 2015-09-08 | 2022-09-01 | Fujifilm Cellular Dynamics | Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells |
ES2903442T3 (en) | 2015-09-08 | 2022-04-01 | Fujifilm Cellular Dynamics Inc | MACS-based purification of stem cell-derived retinal pigment epithelium |
CN105219729B (en) * | 2015-09-28 | 2018-09-25 | 首都医科大学宣武医院 | A kind of method and application thereof using nonconformity plasmid vector induced nerve stem cells |
WO2017070337A1 (en) | 2015-10-20 | 2017-04-27 | Cellular Dynamics International, Inc. | Methods for directed differentiation of pluripotent stem cells to immune cells |
US11352605B2 (en) | 2016-05-12 | 2022-06-07 | Erasmus University Medical Center Rotterdam | Method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium |
CN105861447B (en) * | 2016-06-13 | 2017-12-19 | 广州市搏克生物技术有限公司 | A kind of non-viral iPSCs inducing compositions and its kit |
US20180030478A1 (en) | 2016-07-01 | 2018-02-01 | Research Development Foundation | Elimination of proliferating cells from stem cell-derived grafts |
EP3491134B1 (en) | 2016-08-01 | 2023-10-11 | University of Pittsburgh - of The Commonwealth System of Higher Education | Human induced pluripotent stem cells for high efficiency genetic engineering |
IL264439B1 (en) | 2016-08-17 | 2024-04-01 | Factor Bioscience Inc | non-viral, cell-free composition comprising a synthetic messenger RNA (MRNA) encoding a gene-editing protein for use in treating cancer, and a synthetic RNA encoding a gene-editing protein for use in treatment |
CN110050061A (en) | 2016-10-05 | 2019-07-23 | 富士胶片细胞动力公司 | The mature pedigree of generation is induced multi-potent stem cell from what is destroyed with MeCP2 |
US11458225B2 (en) | 2016-11-09 | 2022-10-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | 3D vascularized human ocular tissue for cell therapy and drug discovery |
US11530388B2 (en) | 2017-02-14 | 2022-12-20 | University of Pittsburgh—of the Commonwealth System of Higher Education | Methods of engineering human induced pluripotent stem cells to produce liver tissue |
JP7181219B2 (en) | 2017-04-18 | 2022-11-30 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | antigen-specific immune effector cells |
WO2018232079A1 (en) | 2017-06-14 | 2018-12-20 | Daley George Q | Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells by episomal plasmid gene transfer |
NL2019517B1 (en) | 2017-09-08 | 2019-03-19 | Univ Erasmus Med Ct Rotterdam | New therapy for Pompe disease |
US20190100729A1 (en) | 2017-10-03 | 2019-04-04 | Wallkill BioPharma, Inc. | Treating diabetes with genetically modified beta cells |
JP2021521792A (en) | 2018-04-20 | 2021-08-30 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | Differentiation method of eyeball cells and their use |
WO2020106622A1 (en) | 2018-11-19 | 2020-05-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Biodegradable tissue replacement implant and its use |
WO2020113029A2 (en) | 2018-11-28 | 2020-06-04 | Board Of Regents, The University Of Texas System | Multiplex genome editing of immune cells to enhance functionality and resistance to suppressive environment |
KR20210096648A (en) | 2018-11-29 | 2021-08-05 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Methods and uses thereof for ex vivo expansion of natural killer cells |
US10501404B1 (en) | 2019-07-30 | 2019-12-10 | Factor Bioscience Inc. | Cationic lipids and transfection methods |
AU2021235185A1 (en) | 2020-03-09 | 2022-11-03 | Fujifilm Corporation | Markers specific for pluripotent stem cells, and methods of using the same |
KR20230019453A (en) | 2020-05-29 | 2023-02-08 | 후지필름 셀룰러 다이내믹스, 인코포레이티드 | Double cell aggregates of retinal pigment epithelial cells and photoreceptors and methods of use thereof |
CN116323677A (en) | 2020-05-29 | 2023-06-23 | 富士胶片细胞动力公司 | Retinal pigment epithelium and photoreceptor bilayer and uses thereof |
EP3922431A1 (en) | 2020-06-08 | 2021-12-15 | Erasmus University Medical Center Rotterdam | Method of manufacturing microdevices for lab-on-chip applications |
IL302728A (en) | 2020-11-13 | 2023-07-01 | Catamaran Bio Inc | Genetically modified natural killer cells and methods of use thereof |
US20220162288A1 (en) | 2020-11-25 | 2022-05-26 | Catamaran Bio, Inc. | Cellular therapeutics engineered with signal modulators and methods of use thereof |
JP2024519515A (en) | 2021-04-07 | 2024-05-15 | センチュリー セラピューティクス,インコーポレイテッド | Compositions and methods for generating gamma-delta T cells from induced pluripotent stem cells - Patents.com |
CN117441010A (en) | 2021-04-07 | 2024-01-23 | 世纪治疗股份有限公司 | Compositions and methods for producing alpha-beta T cells from induced pluripotent stem cells |
US20220389436A1 (en) | 2021-05-26 | 2022-12-08 | FUJIFILM Cellular Dynamics, Inc. | Methods to prevent rapid silencing of genes in pluripotent stem cells |
JP2024520424A (en) | 2021-05-28 | 2024-05-24 | ザ ユナイテッド ステイツ オブ アメリカ, アズ リプレゼンテッド バイ ザ セクレタリー, デパートメント オブ ヘルス アンド ヒューマン サービシーズ | Methods for generating macular, central and peripheral retinal pigment epithelial cells |
CA3220433A1 (en) | 2021-05-28 | 2022-12-01 | Arvydas Maminishkis | Biodegradable tissue scaffold with secondary matrix to host weakly adherent cells |
KR20240056604A (en) | 2021-09-13 | 2024-04-30 | 후지필름 셀룰러 다이내믹스, 인코포레이티드 | Method for producing committed cardiac progenitor cells |
WO2023172514A1 (en) | 2022-03-07 | 2023-09-14 | Catamaran Bio, Inc. | Engineered immune cell therapeutics targeted to her2 and methods of use thereof |
WO2023240147A1 (en) | 2022-06-08 | 2023-12-14 | Century Therapeutics, Inc. | Genetically engineered cells expressing cd16 variants and nkg2d and uses thereof |
US20240003871A1 (en) | 2022-06-29 | 2024-01-04 | FUJIFILM Cellular Dynamics, Inc. | Ipsc-derived astrocytes and methods of use thereof |
WO2024073776A1 (en) | 2022-09-30 | 2024-04-04 | FUJIFILM Cellular Dynamics, Inc. | Methods for the production of cardiac fibroblasts |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090191159A1 (en) * | 2007-06-15 | 2009-07-30 | Kazuhiro Sakurada | Multipotent/pluripotent cells and methods |
US20100003757A1 (en) * | 2008-06-04 | 2010-01-07 | Amanda Mack | Methods for the production of ips cells using non-viral approach |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9223084D0 (en) | 1992-11-04 | 1992-12-16 | Imp Cancer Res Tech | Compounds to target cells |
US5925333A (en) * | 1995-11-15 | 1999-07-20 | Massachusetts Institute Of Technology | Methods for modulation of lipid uptake |
ATE514772T1 (en) * | 1999-08-05 | 2011-07-15 | Abt Holding Co | MULTIPOTENT ADULT STEM CELLS AND METHOD FOR ISOLATION THEREOF |
GB2414480B (en) | 2003-02-07 | 2007-06-27 | Wisconsin Alumni Res Found | Directed genetic modifications of human stem cells |
US8278104B2 (en) * | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US8440461B2 (en) * | 2007-03-23 | 2013-05-14 | Wisconsin Alumni Research Foundation | Reprogramming somatic cells using retroviral vectors comprising Oct-4 and Sox2 genes |
EP2072618A1 (en) * | 2007-12-14 | 2009-06-24 | Johannes Gutenberg-Universität Mainz | Use of RNA for reprogramming somatic cells |
CN101250502A (en) * | 2008-04-01 | 2008-08-27 | 中国科学院上海生命科学研究院 | Method for preparing evoked pluripotent stem cell |
JP5346925B2 (en) | 2008-05-02 | 2013-11-20 | 国立大学法人京都大学 | Nuclear initialization method |
JP2011525794A (en) * | 2008-06-26 | 2011-09-29 | 国立大学法人大阪大学 | iPS cell production method and production kit |
WO2010012077A1 (en) | 2008-07-28 | 2010-02-04 | Mount Sinai Hospital | Compositions, methods and kits for reprogramming somatic cells |
CN102239249A (en) | 2008-10-24 | 2011-11-09 | 威斯康星校友研究基金会 | Pluripotent stem cells obtained by non-viral reprogramming |
-
2009
- 2009-10-23 CN CN2009801480130A patent/CN102239249A/en active Pending
- 2009-10-23 CN CN201610213440.4A patent/CN105802917A/en active Pending
- 2009-10-23 EP EP18200217.0A patent/EP3450545B1/en active Active
- 2009-10-23 JP JP2011533384A patent/JP2012506702A/en not_active Withdrawn
- 2009-10-23 EP EP09744285.9A patent/EP2356221B1/en active Active
- 2009-10-23 US US12/605,220 patent/US8268620B2/en active Active
- 2009-10-23 SG SG10201600234PA patent/SG10201600234PA/en unknown
- 2009-10-23 ES ES18200217T patent/ES2959327T3/en active Active
- 2009-10-23 CA CA2741090A patent/CA2741090C/en active Active
- 2009-10-23 DK DK18200217.0T patent/DK3450545T3/en active
- 2009-10-23 WO PCT/US2009/061935 patent/WO2010048567A1/en active Application Filing
- 2009-10-23 DK DK09744285.9T patent/DK2356221T3/en active
-
2011
- 2011-04-17 IL IL212433A patent/IL212433B/en active IP Right Grant
-
2012
- 2012-09-07 US US13/607,072 patent/US20130217117A1/en not_active Abandoned
-
2015
- 2015-08-24 JP JP2015165051A patent/JP6312638B2/en active Active
-
2016
- 2016-08-26 JP JP2016165793A patent/JP2016220686A/en active Pending
-
2018
- 2018-08-23 JP JP2018155983A patent/JP6861189B2/en active Active
- 2018-12-04 US US16/209,722 patent/US20190330654A1/en not_active Abandoned
-
2021
- 2021-03-29 JP JP2021055183A patent/JP7165228B2/en active Active
- 2021-06-21 US US17/352,873 patent/US20220010331A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090191159A1 (en) * | 2007-06-15 | 2009-07-30 | Kazuhiro Sakurada | Multipotent/pluripotent cells and methods |
US20100003757A1 (en) * | 2008-06-04 | 2010-01-07 | Amanda Mack | Methods for the production of ips cells using non-viral approach |
Non-Patent Citations (11)
Title |
---|
Bouabe (Nucleic Acids Res., Feb 11, 2008, Vol. 36, No. 5, e28, pg 1-9) * |
Cowan et al. Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells.Science, 2005, Vol. 309, pp. 1369-1373. * |
Hasegawa (Stem Cells, March 29, 2007, Vol. 25, pg 1707-1712) * |
Mali (Stem Cells, May 29, 2008, Vol. 26, pg 1998-2005) * |
Martini et al. Different Simian Virus 40 Genomic Regions and Sequences Homologous with SV40 Large T Antigen in DNA of Human Brain and Bone Tumors and of Leukocytes from Blood Donors. Cancer, 2002, Vol. 94, pp. 1037-1048. * |
Okita (Science, Nov. 7, 2008, Vol. 322, pg 949-953) * |
Ozert et al. SV40-Mediated Immortalization of Human Fibroblasts. Experimental Gerontology, 1996, Vol. 31, pp. 303-310. * |
Revazova et al. Patient-Specific Stem Cell Lines Derived from Human Parthenogenetic BlastocystsCloning and Stem Cells, 2007, Vol. 9, pp. 432-449. * |
Supplemental Materials for Yu (Science, 2007, Vol. 318, pg 1917-1920) * |
Takahashi (Nov. 30, 2007, Vol. 131, pg 861-872) * |
Thomson et al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science, 1998, Vol. 282, pp. 1145-1147. * |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11898162B2 (en) | 2007-03-23 | 2024-02-13 | Wisconsin Alumni Research Foundation | Reprogramming somatic cells into pluripotent cells using a vector encoding Oct4 and Sox2 |
US9499786B2 (en) | 2007-03-23 | 2016-11-22 | Wisconsin Alumni Research Foundation | Enriched population of human pluripotent cells with Oct-4 and Sox2 integrated into their genome |
US10106772B2 (en) | 2007-03-23 | 2018-10-23 | Wisconsin Alumni Research Foundation | Somatic cell reprogramming |
US9328332B2 (en) | 2008-06-04 | 2016-05-03 | Cellular Dynamics International, Inc. | Methods for the production of IPS cells using non-viral approach |
US9644184B2 (en) | 2008-06-04 | 2017-05-09 | Cellular Dynamics International, Inc. | Methods for the production of IPS cells using Epstein-Barr (EBV)-based reprogramming vectors |
US9175268B2 (en) | 2008-08-12 | 2015-11-03 | Cellular Dynamics International, Inc. | Methods for the production of iPS cells |
US10738323B2 (en) | 2013-07-12 | 2020-08-11 | Cedars-Sinai Medical Center | Generation of induced pluripotent stem cells from normal human mammary epithelial cells |
US11203758B2 (en) | 2014-10-31 | 2021-12-21 | The Trustees Of The University Of Pennsylvania | Altering gene expression in modified T cells and uses thereof |
KR20170074245A (en) * | 2014-10-31 | 2017-06-29 | 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 | Altering gene expression in modified t cells and uses thereof |
US11208661B2 (en) | 2014-10-31 | 2021-12-28 | The Trustees Of The University Of Pennsylvania | Altering gene expression in modified T cells and uses thereof |
KR102546296B1 (en) * | 2014-10-31 | 2023-06-21 | 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 | Altering gene expression in modified t cells and uses thereof |
WO2016069282A1 (en) * | 2014-10-31 | 2016-05-06 | The Trustees Of The University Of Pennsylvania | Altering gene expression in modified t cells and uses thereof |
US11464182B2 (en) | 2015-07-02 | 2022-10-11 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Method of inducing genetic recombination, and use therefor |
US10221395B2 (en) | 2016-06-16 | 2019-03-05 | Cedars-Sinai Medical Center | Efficient method for reprogramming blood to induced pluripotent stem cells |
US10745671B2 (en) | 2016-06-16 | 2020-08-18 | Cedars-Sinai Medical Center | Efficient method for reprogramming blood to induced pluripotent stem cells |
US11572545B2 (en) | 2016-06-16 | 2023-02-07 | Cedars-Sinai Medical Center | Efficient method for reprogramming blood to induced pluripotent stem cells |
US11970714B2 (en) | 2016-06-16 | 2024-04-30 | Cedars-Sinai Medical Center | Method for reprogramming blood to induced pluripotent stem cells |
US10760057B2 (en) | 2017-07-06 | 2020-09-01 | Wisconsin Alumni Research Foundation | Human pluripotent stem cell-based screening for smooth muscle cell differentiation and disease |
WO2020051453A1 (en) | 2018-09-07 | 2020-03-12 | Wisconsin Alumni Research Foundation | Generation of hematopoietic progenitor cells from human pluripotent stem cells |
Also Published As
Publication number | Publication date |
---|---|
CA2741090A1 (en) | 2010-04-29 |
IL212433A0 (en) | 2011-06-30 |
JP2021094040A (en) | 2021-06-24 |
CN105802917A (en) | 2016-07-27 |
ES2959327T3 (en) | 2024-02-23 |
EP3450545A1 (en) | 2019-03-06 |
US8268620B2 (en) | 2012-09-18 |
DK3450545T3 (en) | 2023-10-02 |
EP3450545B1 (en) | 2023-08-23 |
US20220010331A1 (en) | 2022-01-13 |
JP2018174945A (en) | 2018-11-15 |
JP2012506702A (en) | 2012-03-22 |
SG10201600234PA (en) | 2016-02-26 |
IL212433B (en) | 2019-08-29 |
JP7165228B2 (en) | 2022-11-02 |
US20190330654A1 (en) | 2019-10-31 |
JP2016220686A (en) | 2016-12-28 |
JP6861189B2 (en) | 2021-04-21 |
DK2356221T3 (en) | 2019-02-18 |
CN102239249A (en) | 2011-11-09 |
JP2015213522A (en) | 2015-12-03 |
US20100184227A1 (en) | 2010-07-22 |
WO2010048567A1 (en) | 2010-04-29 |
EP2356221B1 (en) | 2018-11-21 |
EP2356221A1 (en) | 2011-08-17 |
CA2741090C (en) | 2018-10-16 |
JP6312638B2 (en) | 2018-04-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220010331A1 (en) | Pluripotent stem cells obtained by non-viral reporgramming | |
US11898162B2 (en) | Reprogramming somatic cells into pluripotent cells using a vector encoding Oct4 and Sox2 | |
Eminli et al. | Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression | |
EP2476750A1 (en) | Somatic cell reprogramming | |
WO2013177228A1 (en) | Generation of integration/transgene-free stem cells | |
Pfaff et al. | Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells | |
AU2013267048B2 (en) | Somatic cell reprogramming | |
AU2016200360B2 (en) | Somatic cell reprogramming | |
MAHMAUD | GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS USING POLYCISTRONIC LENTIVIRAL VECTOR IN FEEDER-AND SERUM-FREE CULTURE |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: WISCONSIN ALUMNI RESEARCH FOUNDATION, WISCONSIN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:THOMSON, JAMES;YU, JUNYING;SIGNING DATES FROM 20100111 TO 20100112;REEL/FRAME:029076/0617 |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF Free format text: CONFIRMATORY LICENSE;ASSIGNOR:WISCONSIN ALUMNI RESEARCH FOUNDATION;REEL/FRAME:029151/0712 Effective date: 20121004 |
|
STCB | Information on status: application discontinuation |
Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION |