The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

Retinoic acid (RA) induces proliferation arrest, differentiation, and apoptosis, and defects in retinoic acid receptor (RAR) signaling have been implicated in cancer. The human tumor antigen PRAME is overexpressed in a variety of cancers, but its function has remained unclear. We identify here PRAME as a dominant repressor of RAR signaling. PRAME binds to RAR in the presence of RA, preventing ligand-induced receptor activation and target gene transcription through recruitment of Polycomb proteins. PRAME is present at RAR target promoters and inhibits RA-induced differentiation, growth arrest, and apoptosis. Conversely, knockdown of PRAME expression by RNA interference in RA-resistant human melanoma restores RAR signaling and reinstates sensitivity to the antiproliferative effects of RA in vitro and in vivo. Our data suggest that overexpression of PRAME frequently observed in human cancers confers growth or survival advantages by antagonizing RAR signaling.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer great hope for in vitro modeling of Parkinson's disease (PD), as well as for designing cell-replacement therapies. To realize these opportunities, there is an urgent need to develop efficient protocols for the directed differentiation of hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we use lentiviral vectors to drive the expression of LMX1A, which encodes a transcription factor critical for ventral midbrain identity, specifically in neural progenitor cells. We show that clonal lines of hESC engineered to contain one or two copies of this lentiviral vector retain long-term self-renewing ability and pluripotent differentiation capacity. Greater than 60% of all neurons generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9 subtype, compared with ∼10% in green fluorescent protein-engineered controls, as judged by specific marker expression and functional analyses. Moreover, DA neuron precursors differentiated from LMX1A-engineered hESC were able to survive and differentiate when grafted into the brain of adult mice. Finally, we provide evidence that LMX1A overexpression similarly increases the yield of DA neuron differentiation from human iPSC. Taken together, our data show that stable genetic engineering of hESC/iPSC with lentiviral vectors driving controlled expression of LMX1A is an efficient way to generate enriched populations of human A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD and may be helpful for designing future cell-replacement strategies.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of differentiation into any cell type of the organism (pluripotency) and indefinite selfrenewal. Here, we show that the Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss-and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. We show that Rem2 mediates the fibroblastic growth factor 2 (FGF2) signaling pathway to maintain proliferation of hESCs. We demonstrate that Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D 1 to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, we also show that it is as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D 1 expression/localization and suppression of p53 transcription. We show that the effects of Rem2 on cyclin D 1 are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. Our studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use.[Keywords: Rem2; cyclin D1; p53; reprogramming; self-renewal] Supplemental material is available at http://www.genesdev.org. Received October 20, 2009; revised version accepted February 3, 2010. In recent years, the field of pluripotent human embryonic stem cells (hESCs), including the discovery of induced pluripotent stem cells (iPSCs), has moved rapidly in the direction of finding a safe application for clinical use, such as cell replacement therapy and modeling for drug discovery. However, relatively little has been done to advance our mechanistic insights into the properties of self-renewing hESCs, and even less is known about the mechanisms governing iPSC formation. A better understanding of the molecular mechanisms controlling pluripotency and self-renewal would be essential for the clinical translation of hESCs and iPSCs.hESCs were first derived from the pluripotent cells of the blastocyst inner cell mass and can be maintained in vitro indefinitely with the addition of fibroblastic growth factor 2 (FGF2) and other unknown factors secreted from feeder cell layers (Thomson et al. 1998). The pluripotency of hESCs is regulated by a set of unique transcription factors including Oct4, Sox2, and Nanog (Chambers and Smith 2004). It has been shown that a combination of three or four factors of Oct4, Sox2, and KLf4, with or without Myc, can reprogram somatic cells to generate iPSCs (Takahashi and Yamanaka 2006;Takahashi et al. 2007). Analysis of partially reprogrammed iPSCs reveals temporal and separable contributions of the four factors and indicates that ectopic c-Myc ac...

To examine a potential role for phytoestrogens in postmenopausal bone loss, the oophorectomized (OOX) rat model has been used in three studies to investigate the effects of the phytoestrogens coumestrol, zearalanol and a mixture of isoflavones on estrogen-dependent bone loss. In the studies of coumestrol and zearalanol, the rats were allocated to a control group, a phytoestrogen-treated group (1.5 micromol coumestrol or 3.1 mmol zearalanol twice per week, intramuscular) or, in the coumestrol study, an estrogen-treated group (28.1 nmol, intramuscular). In the isoflavone study, the rats were allocated to a control group, an estrogen treated group or a treatment group that received 131.25 mg of phytoestrogens per week incorporated into the nonpurified rat diet. Bone mineral density was measured globally and at the spine and femur at base line and 6 wk post-oophorectomy. In the coumestrol study, blood and urine samples were collected. Compared with the control group, rats receiving coumestrol and zearalanol had significantly reduced bone loss at all sites measured. The estrogen-treated group had significantly greater bone density than the control and the coumestrol-treated groups in the spine and global measurements. Coumestrol reduced urine calcium excretion and the bone resorption markers pyridinoline and deoxypyridinoline after 1 wk of treatment. Oral isoflavone phytoestrogens had no effect on oophorectomized rats including bone loss at the dose used. Thus, for the first time, the bioactivity of coumestrol and zearalanol in preventing bone loss has been demonstrated in a well-recognized model of postmenopausal bone loss.