Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells.

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Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34 + CD43 + hematopoietic progenitors and CD31 + CD43 − endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM-CFCs. hiPSCderived CD43 + cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43 + CD235a + CD41a +/− (erythro-megakaryopoietic), lin − CD34 + CD43 + CD45 − (multi-potent) and lin − CD34 + CD43 + CD45 + (myeloid-skewed) cells. While we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. Materials and methods Cell linesH1 (NIH code WA01), H7 (WA07), H9 (WA09), H13 (WA13), and H14 (WA14) hES cells (passages 27-45; WiCell Research Institute, Madison, WI) were maintained as undifferentiated cells in cocultures with mouse embryonic fibroblasts (MEFs) [11]. iPS(IMR90)-4 and iPS (foreskin)-1 hiPSC lines were obtained by reprogramming IMR90 fetal and newborn foreskin fibroblasts with POU5F1, SOX2, and NANOG; iPS(IMR90)-1, iPS(foreskin)-2, were obtained by reprogramming of fetal and newborn foreskin fibroblasts (ATCC) with POU5F1, SOX2, NANOG, and LIN28 as described [7]. iPS(SK46)-M3-6, iPS(SK46)-M4-8, and iPS(SK46)-M4-10 hiPSC lines were obtained by reprogramming adult skin fibroblasts with POU5F1, SOX2, and NANOG (M3-6) or POU5F1, SOX2, NANOG, Hematopoietic and endothelial differentiation of hiPSCs and hESCs was induced by transferring the cells onto OP9 feeders, as we previously described in details [12,13]. Briefly, undifferentiated hESC/iPSCs were harvested by treatment with 1 mg/ml collagenase IV (Invitrogen) and added to OP9 cultures at an approximate density of 1.2×10 6 /20 ml per 10 cm dish in α-MEM supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah) and 100 µM monothioglycerol (...

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34+CD43+ hematopoietic progenitors and CD31+CD43− endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM-CFCs. hiPSC-derived CD43+ cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43+CD235a+CD41a+/− (erythro-megakaryopoietic), lin−CD34+CD43+CD45− (multi-potent) and lin−CD34+CD43+CD45+ (myeloid-skewed) cells. While we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.

The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.

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