Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed Multidimensional Protein Identification Technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2-associated proteins indicates that they participate in a wide range of processes. Equally important, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4 and Essrb. Moreover, we examined the impact of manipulating the expression of a Sox2-associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC, and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency-associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC.

microRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, have been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.

Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma cells that are associated with the transcription factors Sox2 and Oct-3/4. Other studies have shown that Sox2 and Oct-3/4 work together cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte‐like cells provides a platform to study the molecular basis of human hepatocyte differentiation, to develop cell culture models of liver disease, and to potentially provide hepatocytes for treatment of end‐stage liver disease. Additionally, hepatocyte‐like cells generated from human pluripotent stem cells could serve as platforms for drug discovery, determination of pharmaceutical‐induced hepatotoxicity, and evaluation of idiosyncratic drug‐drug interactions. Here, we describe a step‐wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte‐like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. Curr. Protoc. Stem Cell Biol. 26:1G.4.1‐1G.4.13. © 2013 by John Wiley & Sons, Inc.