Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) with the introduction of Oct4, Sox2, Klf4, and c-Myc. Among these four factors, Oct4 is critical in inducing pluripotency because no transcription factor can substitute for Oct4, whereas Sox2, Klf4, and c-Myc can be replaced by other factors. Here we show that the orphan nuclear receptor Nr5a2 (also known as Lrh-1) can replace Oct4 in the derivation of iPSCs from mouse somatic cells, and it can also enhance reprogramming efficiency. Sumoylation mutants of Nr5a2 with enhanced transcriptional activity can further increase reprogramming efficiency. Genome-wide location analysis reveals that Nr5a2 shares many common gene targets with Sox2 and Klf4, which suggests that the transcription factor trio works in concert to mediate reprogramming. We also show that Nr5a2 works in part through activating Nanog. Together, we show that unrelated transcription factors can replace Oct4 and uncovers an exogenous Oct4-free reprogramming code.

The dominant effect of transcription factors in imparting expanded potency is best exemplified by the reprogramming of fibroblasts to pluripotent cells using retrovirus-mediated transduction of defined transcription factors. In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that have many characteristics of embryonic stem (ES) cells. Here we show that the orphan nuclear receptor Esrrb functions in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb-reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimaeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in self-renewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the upregulation of ES-cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) can be achieved by viral-mediated transduction of defined transcription factors. Moving toward the eventual goal of clinical application, it is necessary to overcome limitations such as low reprogramming efficiency and genomic alterations due to viral integration. Here, we review recent progress made in the usage of genetic factors, chemical inhibitors, and signaling molecules that can either replace core reprogramming factors or enhance reprogramming efficiency. Current iPSC studies will provide a paradigm for the combinatorial use of genetic factors and chemicals for the broader applications to alter cellular states of potency.

The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells.