Control of the Embryonic Stem Cell State

After completing this article, readers should be able to: 1. List the three cells from which mouse pluripotential stem cells can be derived. 2. List the types of specific cells that have been differentiated from murine embryonic stem cells in laboratory investigations. 3. Delineate the potential methods of using human stem cells to minimize immunologic rejection after transplantation. 4. Describe some of the issues requiring resolution before human stem cells can be used in therapies. The dream of one day being able to provide an unlimited supply of human tissues for transplantation came one step closer 2 years ago when two teams of scientists from Johns Hopkins University and the University of Wisconsin announced the successful derivation of human pluripotential stem cells (PSCs). This research immediately caught the public’s eye because of its enormous impact on transplantation therapies and the sources of tissues. Human stem cells are renewable in culture and are capable of differentiating into a wide variety of tissue types. The unlimited ability to divide and the capability to form into almost every cell type provide the source of replacement cells for transplantation and raise the hopes of numerous patients who have debilitating conditions, such as Parkinson disease, Alzheimer disease, stroke, and type I diabetes. Human stem cells will be important for in vitro studies of human gene discovery, for pharmaceutical research such as drug toxicology studies for screening and testing, and as a renewable source of cells for tissue transplantation and gene therapies. In addition to clinical applications, human stem cells provide a powerful tool for biomedical research into human embryogenesis, specific gene functions, and lineage development. PSCs, primarily embryonic stem (ES) cells, have been used extensively in studies of embryogenesis, gene function, and development in the mouse. Present in the early stages of embryo development, PSCs can generate all of …

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Germ layer induction from embryonic stem cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.

GRHL2-Dependent Enhancer Switching Maintains a Pluripotent Stem Cell Transcriptional Subnetwork after Exit from Naive Pluripotency

Role of the Murine Reprogramming Factors in the Induction of Pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Heterogeneity of Embryonic and Adult Stem Cells

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

DOAJ is a unique and extensive index of diverse open access journals from around the world, driven by a growing community, committed to ensuring quality content is freely available online for everyone.

Embryonic stem (ES) cells can grow rapidly and permanently while maintaining their differentiation capacity. To gain insight into how the cell cycle progression of undifferentiated murine ES cells is regulated, we have examined the expression patterns of various replication and cell cycle regulators. Most factors including cyclins, Cdc6, and geminin are rather constitutively expressed during the cell cycle of ES cells. Furthermore, the transcript levels of almost all the cell cycle regulators we investigated except for p21 and p27 are higher in undifferentiated ES cells than in murine embryonic fibroblasts (MEFs), and the increased stability of mRNA in ES cells may be partially responsible for this at least with some of the factors. More strikingly, the transcriptional levels of these factors are strongly correlated with the acetylated state of histone H3 at their promoter regions. However, the methylation state of histone or CpG methylation of the promoter region is not generally correlated significantly with the expression pattern of these factors in both cell types. On the protein level, Cdc6, ASK, cyclin A2, and cyclin B1 are extremely abundant in ES cells compared with MEFs. Furthermore, they are rapidly down-regulated upon induction of differentiation of ES cells. The significance of these findings is discussed in relation to the unusual proliferative properties of ES cells in an undifferentiated state.

Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells