The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Summary The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem cell derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.

generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic b-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing b-cells, rather than pluripotent stem cells, are the major source of new b-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated b-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in b-cell replenishment.The literature on pancreatic b-cells and islets of Langerhans is replete with studies suggesting various mechanisms for b-cell homeostasis and regenerative repair. Early studies on patterns of [ 3 H]thymidine incorporation indicated that adult pancreatic endocrine cells belong to a class of tissues that could be maintained by the self-duplication of differentiated cells [1][2][3] . More recent immunohistochemical observations suggest a stem-cell origin for islet cells, including insulin-expressing b-cells 4 . It has been proposed that these adult pancreatic stem or progenitor cells reside in the epithelium of pancreatic ducts 5,6 , inside islets 7 or in the bone marrow 8 . Others have suggested that b-cells form in the adult by transdifferentiation of pancreatic acinar cells 9 , islet cells that express hormones other than insulin 10 , or splenocytes 11 . In addition to explaining the formation of new b-cells within existing islets, it has also been suggested that whole new islets form (islet neogenesis) by clustering of new b-cells that are derived from stem cells 5,12,13 . However, all of these models and suggestions are, for the most part, based on the interpretation of static histological data rather than direct lineage analysis 14 .We developed a simple method for distinguishing stem-cellderived b-cells from the progeny of pre-existing b-cells. Fully differentiated b-cells, defined here as post-natal cells transcribing the insulin gene, are heritably labelled in transgenic mice with a tamoxifen-inducible Cre/lox system ('pulse'). The label is the expression of the human alkaline phosphatase protein, which can be detected by a histochemical stain. After a long period, during which turnover occurs ('chase'), b-cells are examined for the presence of the label. Cells generated after the pulse are labelled if and only if they are the progeny of pre-existing (labelled) b-cells; new b-cells derived from any non-b source, including stem cells, are not labelled. Differ...

Lithium, one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder, also has dramatic effects on morphogenesis in the early development of numerous organisms. How lithium exerts these diverse effects is unclear, but the favored hypothesis is that lithium acts through inhibition of inositol monophosphatase (IMPase). We show here that complete inhibition of IMPase has no effect on the morphogenesis of Xenopus embryos and present a different hypothesis to explain the broad action of lithium. Our results suggest that lithium acts through inhibition of glycogen synthase kinase-3j3 (GSK-313), which regulates cell fate determination in diverse organisms including Dictyostelium, Drosophila, and Xenopus. Lithium potently inhibits GSK-313 activity (Ki = 2 mM), but is not a general inhibitor of other protein kinases. In support of this hypothesis, lithium treatment phenocopies loss of GSK-3j8 function in Xenopus and Dictyostelium. These observations help explain the effect of lithium on cell-fate determination and could provide insights into the pathogenesis and treatment ofbipolar disorder.

One goal of regenerative medicine is to instructively convert adult cells into other cell types for tissue repair and regeneration. Although isolated examples of adult cell reprogramming are known, there is no general understanding of how to turn one cell type into another in a controlled manner. Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble beta-cells. The induced beta-cells are indistinguishable from endogenous islet beta-cells in size, shape and ultrastructure. They express genes essential for beta-cell function and can ameliorate hyperglycaemia by remodelling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.

Summary Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.