title: Generation of human induced pluripotent stem cells using epigenetic regulators reveals a germ cell-like identity in partially reprogrammed colonies.
abstract: Previous studies have shown that induced pluripotent stem cells (iPSCs) can be derived from fibroblasts by ectopic expression of four transcription factors, OCT4, SOX2, KLF4 and c-MYC using various methods. More recent studies have focused on identifying alternative approaches and factors that can be used to increase reprogramming efficiency of fibroblasts to pluripotency. Here, we use nucleofection, morpholino technologies and novel epigenetic factors, which were chosen based on their expression profile in human embryos, fibroblasts and undifferentiated/differentiated human embryonic stem cells (hESCs) and conventionally generated iPSCs, to reprogram human fibroblasts into iPSCs. By over expressing DNMT3B, AURKB, PRMT5 and/or silencing SETD7 in human fibroblasts with and without NANOG, hTERT and/or SV40 overexpression, we observed the formation of colonies resembling iPSCs that were positive for certain pluripotency markers, but exhibited minimal proliferation. More importantly, we also demonstrate that these partially-reprogrammed colonies express high levels of early to mid germ cell-specific genes regardless of the transfection approach, which suggests conversion to a germ cell-like identity is associated with early reprogramming. These findings may provide an additional means to evaluate human germ cell differentiation in vitro, particularly in the context of pluripotent stem cell-derived germ cell development, and contribute to our understanding of the epigenetic requirements of the reprogramming process.
authors.affiliation: Center for Reproductive and Stem Cell Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California, United States of America ; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Akshi
authors.initials: A
authors.lastname: Goyal
authors.affiliation: Center for Reproductive and Stem Cell Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California, United States of America ; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Shawn L
authors.initials: SL
authors.lastname: Chavez
authors.affiliation: Center for Reproductive and Stem Cell Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California, United States of America ; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Renee A
authors.initials: RA
authors.lastname: Reijo Pera
mesh_terms: D064107:Aurora Kinase B / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D065150:Cellular Reprogramming / Q000187:drug effects; D016000:Cluster Analysis; D003114:Colony-Forming Units Assay; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D000071317:Nanog Homeobox Protein; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014635:Valproic Acid / Q000494:pharmacology; D000097584:DNA Methyltransferase 3B
chemical_list: D064107:Aurora Kinase B; D001374:Azacitidine; D004248:DNA (Cytosine-5-)-Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones; D018398:Homeodomain Proteins; D000090062:Kruppel-Like Factor 4; D000071317:Nanog Homeobox Protein; D011484:Protein-Arginine N-Methyltransferases; D019098:Telomerase; D014157:Transcription Factors; D014635:Valproic Acid; D000097584:DNA Methyltransferase 3B; C000715467:KLF4 protein, human; C486780:NANOG protein, human; C400529:PRMT5 protein, human; C540312:SETD7 protein, human