US20220010331A1 - Pluripotent stem cells obtained by non-viral reporgramming - Google Patents
Pluripotent stem cells obtained by non-viral reporgramming Download PDFInfo
- Publication number
- US20220010331A1 US20220010331A1 US17/352,873 US202117352873A US2022010331A1 US 20220010331 A1 US20220010331 A1 US 20220010331A1 US 202117352873 A US202117352873 A US 202117352873A US 2022010331 A1 US2022010331 A1 US 2022010331A1
- Authority
- US
- United States
- Prior art keywords
- pep4
- cells
- reprogramming
- cell
- human
- 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.)
- Pending
Links
- 230000003612 virological effect Effects 0.000 title description 4
- 210000001778 pluripotent stem cell Anatomy 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 134
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 26
- 241000288906 Primates Species 0.000 claims abstract description 21
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 claims description 15
- 102100024270 Transcription factor SOX-2 Human genes 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 15
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 14
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 14
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 14
- 108700026244 Open Reading Frames Proteins 0.000 claims description 11
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 claims description 8
- 102100020677 Krueppel-like factor 4 Human genes 0.000 claims description 8
- 239000000427 antigen Substances 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 101000984042 Homo sapiens Protein lin-28 homolog A Proteins 0.000 claims description 6
- 102100025460 Protein lin-28 homolog A Human genes 0.000 claims description 6
- 108091026890 Coding region Proteins 0.000 claims description 4
- -1 NANOG Proteins 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 239000013598 vector Substances 0.000 abstract description 65
- 230000008672 reprogramming Effects 0.000 abstract description 54
- 241000700605 Viruses Species 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 4
- 230000002458 infectious effect Effects 0.000 abstract description 4
- 230000014509 gene expression Effects 0.000 description 41
- 210000002950 fibroblast Anatomy 0.000 description 29
- 108700019146 Transgenes Proteins 0.000 description 27
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 17
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 17
- 210000003953 foreskin Anatomy 0.000 description 17
- 230000000694 effects Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 210000000130 stem cell Anatomy 0.000 description 8
- 238000001890 transfection Methods 0.000 description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000010361 transduction Methods 0.000 description 5
- 101710128836 Large T antigen Proteins 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 239000003636 conditioned culture medium Substances 0.000 description 4
- 230000001605 fetal effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000000392 somatic effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000002123 temporal effect Effects 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 241001529936 Murinae Species 0.000 description 3
- 238000000339 bright-field microscopy Methods 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 238000009795 derivation Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 210000001654 germ layer Anatomy 0.000 description 3
- 230000004660 morphological change Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000005014 ectopic expression Effects 0.000 description 2
- 230000002500 effect on skin Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002894 multi-fate stem cell Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 101000693916 Gallus gallus Albumin Proteins 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101000655352 Homo sapiens Telomerase reverse transcriptase Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101100494726 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pep-4 gene Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 241001068263 Replication competent viruses Species 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 206010043276 Teratoma Diseases 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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
- 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)).
- FIGS. 1A-1B illustrate the effect on reprogramming efficiency of different nucleotide sequences that link transgenes on the vector(s) delivered during the reprogramming methods.
- 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.
- 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.
- 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.
- 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).
- 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 6-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 6-mercaptoethanol and 100 ng/ml zebrafish basic fibroblast growth factor (zbFGF) as previously described
- 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. 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.
- 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)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Transplantation (AREA)
- Developmental Biology & Embryology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- This application is a continuation of U.S. patent application Ser. No. 16/209,722, filed Dec. 4, 2018, which is a continuation of U.S. patent application Ser. No. 13/607,072, filed Sep. 7, 2012, which is a continuation of U.S. patent application Ser. No. 12/605,220, filed Oct. 23, 2009, which issued 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, each of which is incorporated herein by reference as if set forth in its entirety.
- This invention was made with government support under GM081629 and RR000167 awarded by the National Institutes of Health. The government has certain rights in the 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.
-
FIGS. 1A-1B illustrate the effect on reprogramming efficiency of different nucleotide sequences that link transgenes on the vector(s) delivered during the reprogramming methods. -
FIGS. 2A-2C illustrate the effect on reprogramming efficiency of c-Myc, KLF-4, and SV40 large T antigen gene expression in human newborn foreskin fibroblasts. -
FIGS. 3A-3C 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. -
FIGS. 4A-4D illustrate reprogramming of human newborn foreskin fibroblasts with episomal vector-mediated transgene expression. -
FIGS. 5A-5B 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, mesodern and ectoderm). When introduced into an immunocompromised animal, such as a SCID mouse, the phuripotent 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), niTeSR™ (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-O2S” 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:1) 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. IRES1 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-IRES1-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 N4NOG has a profound positive effect on reprogramming efficiency when OCT4, SOA2, 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 ß-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 6-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.
-
TABLE 1 Reprogramming genes and translation elements SEQ Accession # Gene Symbol Abbr. Source ID NO or sequence OCT4 O hESC 1 NM_002701 SOX2 S hESC 2 NM_003106 NANOG N hESC 3 NM_024865 LN28 L hESC 4 NM_024674 c- Myc M hESC 5 NM_002467 KLF4 K hESC 6 NM_004235 SV40 T T pBABE-puro 7 EF579667 SV40 LT p TERT TERT pBABE-hygro- 8 NM_198253 hTERT IRES1 1 pIRESpuro3 — IRES2 2 pIRES2EGFP — F2A F2A (synthesized) 9 CMV C 10 EF1α E 11 EF2α — 12 -
TABLE 2 Episomal constructs # Name Size (bp) 1 pCEP4- EGFP 10984 2 b pEP4-E-O2S(2) 13523 3 b pEP4-E-M2K 14293 4 a pCEP4-M2K 13643 5 b pEP4-E-K2M 14268 6 a pCEP4-K2M 13636 7 b pEP4-E-N2K 13819 8 b pEP4-E-T2K 15071 9 a pCEP4-M2L 12852 10 b pEP4-E-N2L 13020 11 b pEP4-E-T2L 14284 12 c pEP4-E-O2S-C-M2K 16038 13 c pEP4-E-O2S-E-M2K 16680 14 c pEP4-E-O2S-C-K2M 16010 15 c pEP4-E-O2S-E-K2M 16652 16 c pEP4-E-O2S-E-N2K 16206 17 c pEP4-E-O2S-E-T2K 17458 18 c pEP4-E-O2S-E-N2L 15415 19 c pEP4-E-O2S-E-T2L 16679 20 c pEP4-O2S-C-M2L 15247 21 c pEP4-E-O2S-E-K2T 17474 22 c pEP4-E-O2S-C-M2L-E-N2K 19956 23 c pEP4-E-O2S-C-M2K-E-N2L 19956 24 c pEP4-E-O2S-C-K2M-E-N2L 19949 25 c pEP4-E-O2S-C-M2L-E-T2K 21220 26 c pEP4-E-O2S-C-M2K-E-T2L 21220 27 c pEP4-E-O2S-C-K2M-E-T2L 21213 28 c pEP4-E-O2S-C-M2L-E-K2T 21224 a All linked gene cassettes were cloned into the pCEP4-EGFP between BamHI and NheI restriction sites. b All linked gene cassettes plus the EF1α promoter were cloned into the pCEP4-EGFP between BamHI and SpeI (19) restriction sites. c All expression cassettes were cloned into the pCEP4-EGFP between BamHI and NruI restriction sites. -
TABLE 3 Combinations of episomal constructs tested for reprogramming activity Equivalent of pCEP4-EGF(μg) Test # Plasmids μg Morph. Changes AP+ colony/plate EXPERIMENT 1 6.3 1 pEP4-E-O2S-C-M2K 9.2 +/− 0 6.3 2 pEP4-E-O2S-K2Neo 9.3 +/− 0 6.3 pCEP4-M2L 7.4 6.3 3 pEP4-E-O2S-E-N2K 9.3 +/− 0 6.3 pCEP4-M2L 7.4 6.3 4 pEP4-E-O2S-E-T2K 10 +++ 0 6.3 pCEP4-M2L 7.4 6.3 5 pEP4-E-O2S-E-TERT2K 10.8 +/− 0 6.3 pCEP4-M2L 7.4 6.3 6 pEP4-E-O2S-C-M2L 8.7 +/− 0 6.3 pEP4-E-N2K 7.9 6.3 7 pEP4-E-O2S-C-M2L 8.7 + 0 6.3 pEP4-E-T2K 8.6 6.3 8 pEP4-E-O2S-C-M2L 8.7 +/− 0 6.3 pEP4-E-TERT2K 9.4 EXPERIMENT 2 1 pEP4-E-O2S-C-M2K 5.0 +/− 0 3.3 2 pEP4-E-O2S-E-M2K 5.0 +/− 0 3.3 3 pEP4-E-O2S-C-K2M 5.0 +/− 0 3.3 4 pEP4-E-O2S-E-K2M 5.0 +/− 0 2.5 5 pEP4-E-O2S(2) 3.0 +/− 0 2.5 pCEP4-M2K 3.0 2.5 6 pEP4-E-O2S(2) 3.0 +/− 0 2.3 pEP4-E-M2K 3.0 2.5 7 pEP4-E-O2S(2) 3.0 +/− 0 2.5 pCEP4-K2M 3.0 2.5 8 pEP4-E-O2S(2) 3.0 +/− 0 2.3 pEP4-E-K2M 3.0 1.7 9N pEP4-E-O2S(2) 2.0 +/− 0 1.5 pEP4-E-N2K 2.0 1.7 pCEP4-M2L 2.0 +/− 0 1.7 10N pEP4-E-O2S(2) 2.0 +/− 0 1.7 pEP4-E-N2L 2.0 1.7 pCEP4-M2K 2.0 1.7 11N pEP4-E-O2S(2) 2.0 +/− 0 1.7 pEP4-E-N2L 2.0 1.5 pEP4-E-M2K 2.0 1.7 12N pEP4-E-O2S(2) 2.0 +/− 0 1.7 pEP4-E-N2L 2.0 1.7 pCEP4-K2M 2.0 1.7 13N pEP4-E-O2S(2) 2.0 +/− 0 1.7 pEP4-E-N2L 2.0 1.5 pEP4-E-K2M 2.0 2.3 14N pEP4-E-O2S-E-N2K 3.5 +/− 0 2.1 pCEP4-M2L 2.5 2.5 15N pEP4-E-O2S-E-N2L 3.5 +/− 0 2.1 pCEP4-M2K 2.5 2.5 16N pEP4-E-O2S-E-N2L 3.5 +/− 0 1.9 pEP4-E-M2K 2.5 2.5 17N pEP4-E-O2S-E-N2L 3.5 +/− 0 2.1 pCEP4-K2M 2.5 2.5 18N pEP4-E-O2S-E-N2L 3.5 +/− 0 1.9 pEP4-E-K2M 2.5 EXPERIMENT 3 1.7 9T pEP4-E-O2S(2) 2.0 +/− 0 1.4 pEP4-E-T2K 2.0 1.7 pCEP4-M2L 2.0 1.7 10T pEP4-E-O2S(2) 2.0 + 0 1.5 pEP4-E-T2L 2.0 1.7 pCEP4-M2K 2.0 1.7 11T pEP4-E-O2S(2) 2.0 + 0 1.5 pEP4-E-T2L 2.0 1.5 pEP4-E-M2K 2.0 1.7 12T pEP4-E-O2S(2) 2.0 +/− 0 1.5 pEP4-E-T2L 2.0 1.7 pCEP4-K2M 2.0 1.7 13T pEP4-E-O2S(2) 2.0 +/− 0 1.5 pEP4-E-T2L 2.0 1.5 pEP4-E-K2M 2.0 2.2 14T pEP4-E-O2SET2K 3.5 +++ 0 2.1 pCEP4-M2L 2.5 2.3 15T pEP4-E-O2S-E-T2L 3.5 + 0 2.1 pCEP4-M2K 2.5 2.3 16T pEP4-E-O2S-E-T2L 3.5 + 0 1.9 pEP4-E-M2K 2.5 2.3 17T pEP4-E-O2S-E-T2L 3.5 +/− 0 2.1 pCEP4-K2M 2.5 2.3 18T pEP4-E-O2S-E-T2L 3.5 +/− 0 1.9 pEP4-E-K2M 2.5 1.9 19 pEP4-E-O2S-E-T2K 3.0 +++ 1 2.0 pEP4-E-O2S-E-N2K 3.0 1.7 pCEP4-M2L 2.0 EXPERIMENT 4 6 1 pEP4-E-O2S-C-M2K-E-N2L 10.9 +/− 0 4 2 pEP4-E-O2S-C-M2K-E-N2L 7.3 +++ 0 2 pEP4-E-O2S-E-T2K 3.2 6 3 pEP4-E-O2S-C-K2M-E-N2L 10.9 +/− 0 4 4 pEP4-E-O2S-C-K2M-E-N2L 7.3 ++ 2 2 pEP4-E-O2S-E-T2K 3.2 3 5 pEP4-E-O2S-E-N2L 4.2 +/− 0 3 pEP4-E-O2S-E-M2K 4.6 3 6 pEP4-E-O2S-E-N2L 4.2 ++ 1 2 pEP4-E-O2S-E-T2K 3.2 3 pEP4-E-O2S-E-M2K 4.6 3 7 pEP4-E-O2S-E-N2L 4.2 +/− 0 3 pEP4-E-O2S-C-M2K 4.4 3 8 pEP4-E-O2S-E-N2L 4.2 + 0 2 pEP4-E-O2S-E-T2K 3.2 3 pEP4-E-O2S-C-M2K 4.4 3 9 pEP4-E-O2S-E-N2L 4.2 +/− 0 3 pEP4-E-O2S-E-K2M 4.5 3 10 pEP4-E-O2S-E-N2L 4.2 +/− 0 2 pEP4-E-O2S-E-T2K 3.2 3 pEP4-E-O2S-E-K2M 4.5 3 11 pEP4-E-O2S-E-N2L 4.2 +/− 0 3 pEP4-E-O2S-C-K2M 4.4 3 12 pEP4-E-O2S-E-N2L 4.2 + 0 2 pEP4-E-O2S-E-T2K 3.2 3 pEP4-E-O2S-C-K2M 4.4 2 13 pEP4-E-O2S-C-M2L-E-T2K 3.9 + 0 4 pEP4-E-O2S-E-N2K 5.9 6 14 pEP4-E-O2S-C-M2K-E-T2L 11.6 + 0 3 15 pEP4-E-O2S-C-M2K-E-T2L 5.8 + 0 3 pEP4-E-O2S-E-N2K 4.4 6 16 pEP4-E-O2S-C-K2M-E-T2L 11.6 +/− 0 3 17 pEP4-E-O2S-C-K2M-E-T2L 5.8 + 0 3 pEP4-E-O2S-E-N2K 4.4 6 18 pEP4-E-O2S-C-M2L-E-K2T 11.6 +/− 0 3 19 pEP4-E-O2S-C-M2L-E-K2T 5.8 +/− 0 3 pEP4-E-O2S-E-N2K 4.4 3 20 pEP4-E-O2S-E-K2T 4.8 +/− 0 3 pEP4-E-O2S-E-N2K 4.4 2 pEP4-E-O2S-C-M2L 2.8 +/−: No or very few colonies with morphological change were observed (FIG. 4B). +, ++ and +++: Different number (from less to more) of colonies with morphological change were observed. - 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 (6)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/352,873 US20220010331A1 (en) | 2008-10-24 | 2021-06-21 | Pluripotent stem cells obtained by non-viral reporgramming |
Applications Claiming Priority (5)
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 |
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 |
Related Parent 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 |
---|---|
US20220010331A1 true US20220010331A1 (en) | 2022-01-13 |
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 (3)
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 |
Country Status (10)
Country | Link |
---|---|
US (4) | US8268620B2 (en) |
EP (2) | EP3450545B1 (en) |
JP (5) | JP2012506702A (en) |
CN (2) | CN105802917A (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) |
Families Citing this family (78)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8278104B2 (en) | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US20090227032A1 (en) * | 2005-12-13 | 2009-09-10 | Kyoto University | Nuclear reprogramming factor and induced pluripotent stem cells |
US8129187B2 (en) | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
PT1970446E (en) * | 2005-12-13 | 2011-09-01 | Univ Kyoto | Nuclear reprogramming factor |
SG193653A1 (en) | 2007-03-23 | 2013-10-30 | Wisconsin Alumni Res Found | Somatic cell reprogramming |
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 |
KR101661940B1 (en) * | 2008-05-02 | 2016-10-04 | 고쿠리츠 다이가쿠 호진 교토 다이가쿠 | Method of nuclear reprogramming |
ES2587395T3 (en) | 2008-06-04 | 2016-10-24 | Cellular Dynamics International, Inc. | Procedures for the production of IPS cells using a non-viral approach |
JP2012500005A (en) * | 2008-08-12 | 2012-01-05 | セルラー ダイナミクス インターナショナル, インコーポレイテッド | Method for generating iPS cells |
EP3450545B1 (en) * | 2008-10-24 | 2023-08-23 | Wisconsin Alumni Research Foundation | Pluripotent stem cells obtained by non-viral reprogramming |
CN102459575A (en) | 2009-06-05 | 2012-05-16 | 细胞动力国际有限公司 | Reprogramming t cells and hematophietic cells |
US8048675B1 (en) * | 2010-05-12 | 2011-11-01 | Ipierian, Inc. | Integration-free human induced pluripotent stem cells from blood |
WO2011159684A2 (en) | 2010-06-15 | 2011-12-22 | Cellular Dynamics International, Inc. | Generation of induced pluripotent stem cells from small volumes of peripheral blood |
US9279107B2 (en) | 2010-08-05 | 2016-03-08 | Wisconsin Alumni Research Foundation | Simplified basic media for human pluripotent cell culture |
BR112013002811A8 (en) | 2010-08-05 | 2020-01-28 | Wisconsin Alumni Res Found | simplified basic means for human pluripotent cell culture |
US20130296183A1 (en) | 2010-09-17 | 2013-11-07 | President And Fellows Of Harvard College | Functional genomics assay for characterizing pluripotent stem cell utility and safety |
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 |
CA2852244C (en) | 2011-10-17 | 2023-10-17 | Minerva Biotechnologies Corporation | Media for stem cell proliferation 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 |
WO2013086008A1 (en) | 2011-12-05 | 2013-06-13 | Factor Bioscience Inc. | Methods and products for transfecting cells |
US8497124B2 (en) | 2011-12-05 | 2013-07-30 | Factor Bioscience Inc. | Methods and products for reprogramming cells to a less differentiated state |
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 |
EP3786298A1 (en) | 2012-11-01 | 2021-03-03 | Factor Bioscience Inc. | Methods and products for expressing proteins in cells |
US20150368713A1 (en) | 2013-02-01 | 2015-12-24 | THE UNITED STATES OF AMERICAN, as represented by the Secretary, Department of Health and Human Serv | METHOD FOR GENERATING RETINAL PIGMENT EPITHELIUM (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCs) |
US11085067B2 (en) | 2013-06-10 | 2021-08-10 | President And Fellows Of Harvard College | Early developmental genomic assay for characterizing pluripotent stem cell utility and safety |
WO2015006725A2 (en) | 2013-07-12 | 2015-01-15 | Cedars-Sinai Medical Center | Generation of induced pluripotent stem cells from normal human mammary epithelial cells |
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 |
EP3099801B1 (en) | 2014-01-31 | 2020-03-18 | Factor Bioscience Inc. | Synthetic rna for use in the treatment of dystrophic epidermolysis bullosa |
EP3134515B1 (en) | 2014-04-24 | 2019-03-27 | Board of Regents, The University of Texas System | Application of induced pluripotent stem cells to generate adoptive cell therapy products |
KR20240075936A (en) * | 2014-10-31 | 2024-05-29 | 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 | Altering gene expression in cart cells and uses thereof |
US11241505B2 (en) | 2015-02-13 | 2022-02-08 | Factor Bioscience Inc. | Nucleic acid products and methods of administration thereof |
CA2982568A1 (en) * | 2015-04-14 | 2016-10-20 | Kyoto University | Method for producing stem cell clone suitable for inducing differentiation into somatic cells |
JP6449138B2 (en) * | 2015-07-02 | 2019-01-09 | 株式会社豊田中央研究所 | Method for inducing genetic recombination and use thereof |
ES2970537T3 (en) | 2015-09-08 | 2024-05-29 | Us Health | Method for reproducible differentiation of clinical-grade retinal pigment epithelial cells |
LT3347457T (en) | 2015-09-08 | 2022-02-10 | 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 |
CN108350429B (en) | 2015-10-20 | 2022-02-25 | 富士胶片细胞动力公司 | Method for directed differentiation of pluripotent stem cells into immune cells |
WO2017196175A1 (en) | 2016-05-12 | 2017-11-16 | Erasmus University Medical Center Rotterdam | A 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 |
US10221395B2 (en) | 2016-06-16 | 2019-03-05 | 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 |
JP7099967B2 (en) | 2016-07-01 | 2022-07-12 | リサーチ ディベロップメント ファウンデーション | Elimination of Proliferative Cells from Stem Cell-Derived Grafts |
US11866733B2 (en) | 2016-08-01 | 2024-01-09 | University of Pittsburgh—of the Commonwealth System of Higher Education | Human induced pluripotent stem cells for high efficiency genetic engineering |
CA3033788A1 (en) | 2016-08-17 | 2018-02-22 | Factor Bioscience Inc. | Nucleic acid products and methods of administration thereof |
WO2018067826A1 (en) | 2016-10-05 | 2018-04-12 | Cellular Dynamics International, Inc. | Generating mature lineages from induced pluripotent stem cells with mecp2 disruption |
AU2017359330B2 (en) | 2016-11-09 | 2022-03-10 | 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 |
CN110891967A (en) | 2017-04-18 | 2020-03-17 | 富士胶片细胞动力公司 | 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 |
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 |
NL2019517B1 (en) | 2017-09-08 | 2019-03-19 | Univ Erasmus Med Ct Rotterdam | New therapy for Pompe disease |
EP3692139A1 (en) | 2017-10-03 | 2020-08-12 | Wallkill Biopharma, Inc. | Treating diabetes with genetically modified beta cells |
AU2019256723A1 (en) | 2018-04-20 | 2020-11-05 | FUJIFILM Cellular Dynamics, Inc. | Method for differentiation of ocular cells and use thereof |
WO2020051453A1 (en) | 2018-09-07 | 2020-03-12 | Wisconsin Alumni Research Foundation | Generation of hematopoietic progenitor cells from human pluripotent stem cells |
KR20210095885A (en) | 2018-11-19 | 2021-08-03 | 더 유나이티드 스테이츠 오브 어메리카, 애즈 리프리젠티드 바이 더 세크러테리, 디파트먼트 오브 헬쓰 앤드 휴먼 서비씨즈 | Biodegradable tissue replacement implants and uses thereof |
EP3887518A2 (en) | 2018-11-28 | 2021-10-06 | Board of Regents, The University of Texas System | Multiplex genome editing of immune cells to enhance functionality and resistance to suppressive environment |
WO2020112493A1 (en) | 2018-11-29 | 2020-06-04 | Board Of Regents, The University Of Texas System | Methods for ex vivo expansion of natural killer cells and use thereof |
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 |
US20230212509A1 (en) | 2020-05-29 | 2023-07-06 | FUJIFILM Cellular Dynamics, Inc. | Bilayer of retinal pigmented epithelium and photoreceptors and use thereof |
EP4157294A1 (en) | 2020-05-29 | 2023-04-05 | FUJIFILM Cellular Dynamics, Inc. | Retinal pigmented epithelium and photoreceptor dual cell aggregates and methods of use thereof |
EP3922431A1 (en) | 2020-06-08 | 2021-12-15 | Erasmus University Medical Center Rotterdam | Method of manufacturing microdevices for lab-on-chip applications |
WO2022104109A1 (en) | 2020-11-13 | 2022-05-19 | Catamaran Bio, Inc. | Genetically modified natural killer cells and methods of use thereof |
AU2021388155A1 (en) | 2020-11-25 | 2023-06-15 | Catamaran Bio, Inc. | Cellular therapeutics engineered with signal modulators and methods of use thereof |
CA3214045A1 (en) | 2021-04-07 | 2022-10-13 | Century Therapeutics, Inc. | Compositions and methods for generating gamma-delta t cells from induced pluripotent stem cells |
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 |
CN117881777A (en) | 2021-05-26 | 2024-04-12 | 富士胶片细胞动力公司 | Method for preventing rapid gene silencing in pluripotent stem cells |
WO2022251477A1 (en) | 2021-05-28 | 2022-12-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Biodegradable tissue scaffold with secondary matrix to host weakly adherent 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 |
AU2022343749A1 (en) | 2021-09-13 | 2024-03-28 | FUJIFILM Cellular Dynamics, Inc. | Methods for the production of 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 |
WO2024006911A1 (en) | 2022-06-29 | 2024-01-04 | FUJIFILM Holdings America Corporation | 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 (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009006930A1 (en) * | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc. | Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue |
US20100003757A1 (en) * | 2008-06-04 | 2010-01-07 | Amanda Mack | Methods for the production of ips cells using non-viral approach |
US8183038B2 (en) * | 2007-03-23 | 2012-05-22 | Wisconsin Alumni Research Foundation | Composition comprising recombinant nucleic acid encoding Sox2, Oct-4, Nanog and Lin28 |
Family Cites Families (11)
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 |
CA2852534A1 (en) * | 1999-08-05 | 2001-02-15 | Abt Holding Company | Multipotent adult stem cells and methods for isolation |
US20060128018A1 (en) | 2003-02-07 | 2006-06-15 | Zwaka Thomas P | 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 |
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 |
KR101661940B1 (en) | 2008-05-02 | 2016-10-04 | 고쿠리츠 다이가쿠 호진 교토 다이가쿠 | Method of nuclear reprogramming |
WO2009157201A1 (en) * | 2008-06-26 | 2009-12-30 | Osaka University | Method and kit for preparing ips cells |
WO2010012077A1 (en) * | 2008-07-28 | 2010-02-04 | Mount Sinai Hospital | Compositions, methods and kits for reprogramming somatic cells |
EP3450545B1 (en) * | 2008-10-24 | 2023-08-23 | Wisconsin Alumni Research Foundation | Pluripotent stem cells obtained by non-viral reprogramming |
-
2009
- 2009-10-23 EP EP18200217.0A patent/EP3450545B1/en active Active
- 2009-10-23 ES ES18200217T patent/ES2959327T3/en active Active
- 2009-10-23 US US12/605,220 patent/US8268620B2/en active Active
- 2009-10-23 CN CN201610213440.4A patent/CN105802917A/en active Pending
- 2009-10-23 JP JP2011533384A patent/JP2012506702A/en not_active Withdrawn
- 2009-10-23 WO PCT/US2009/061935 patent/WO2010048567A1/en active Application Filing
- 2009-10-23 CA CA2741090A patent/CA2741090C/en active Active
- 2009-10-23 SG SG10201600234PA patent/SG10201600234PA/en unknown
- 2009-10-23 DK DK18200217.0T patent/DK3450545T3/en active
- 2009-10-23 DK DK09744285.9T patent/DK2356221T3/en active
- 2009-10-23 EP EP09744285.9A patent/EP2356221B1/en active Active
- 2009-10-23 CN CN2009801480130A patent/CN102239249A/en active Pending
-
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 (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8183038B2 (en) * | 2007-03-23 | 2012-05-22 | Wisconsin Alumni Research Foundation | Composition comprising recombinant nucleic acid encoding Sox2, Oct-4, Nanog and Lin28 |
US8440461B2 (en) * | 2007-03-23 | 2013-05-14 | Wisconsin Alumni Research Foundation | Reprogramming somatic cells using retroviral vectors comprising Oct-4 and Sox2 genes |
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 |
WO2009006930A1 (en) * | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc. | Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue |
US20100003757A1 (en) * | 2008-06-04 | 2010-01-07 | Amanda Mack | Methods for the production of ips cells using non-viral approach |
Non-Patent Citations (1)
Title |
---|
Thomson (Science, 1998, Vol. 282, pp. 1145-1147) (Year: 1998) * |
Also Published As
Publication number | Publication date |
---|---|
JP2015213522A (en) | 2015-12-03 |
EP2356221A1 (en) | 2011-08-17 |
US20100184227A1 (en) | 2010-07-22 |
CA2741090A1 (en) | 2010-04-29 |
DK3450545T3 (en) | 2023-10-02 |
JP2021094040A (en) | 2021-06-24 |
JP2016220686A (en) | 2016-12-28 |
DK2356221T3 (en) | 2019-02-18 |
EP3450545A1 (en) | 2019-03-06 |
JP6861189B2 (en) | 2021-04-21 |
WO2010048567A1 (en) | 2010-04-29 |
JP7165228B2 (en) | 2022-11-02 |
JP2018174945A (en) | 2018-11-15 |
CA2741090C (en) | 2018-10-16 |
US20190330654A1 (en) | 2019-10-31 |
US8268620B2 (en) | 2012-09-18 |
SG10201600234PA (en) | 2016-02-26 |
IL212433A0 (en) | 2011-06-30 |
IL212433B (en) | 2019-08-29 |
JP2012506702A (en) | 2012-03-22 |
ES2959327T3 (en) | 2024-02-23 |
CN105802917A (en) | 2016-07-27 |
EP3450545B1 (en) | 2023-08-23 |
US20130217117A1 (en) | 2013-08-22 |
EP2356221B1 (en) | 2018-11-21 |
JP6312638B2 (en) | 2018-04-18 |
CN102239249A (en) | 2011-11-09 |
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 | |
Pfaff et al. | Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells | |
US20130065814A1 (en) | Inductive production of pluripotent stem cells using synthetic transcription factors | |
AU2013267048B2 (en) | Somatic cell reprogramming | |
MAHMAUD | GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS USING POLYCISTRONIC LENTIVIRAL VECTOR IN FEEDER-AND SERUM-FREE CULTURE | |
AU2016200360A1 (en) | Somatic cell reprogramming |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
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:058035/0034 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |