Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1

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.

P-1017: Using mouse blastocyst as a reprogramming vector for human dental pulp stem cells

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency. Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG's actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency. Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process.Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF).Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression.Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation.Conclusions: We propose that ZMYM2 curtails NANOG’s actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

Role of the Murine Reprogramming Factors in the Induction of Pluripotency

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

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

recent Nobel Prize in medicine was awarded to two stemcell researchers, John Gurdon and Shinya Yamanaka, for theirachievements in stem cell research and reprogramming ofsomatic cells. Flow cytometry is by nature the ideal tool toidentify, characterize, and isolate stem and progenitor cells forresearch and potential clinical use (1). The major strength offlow cytometry is its ability to rapidly perform highlymultiplexed quantitative measurements on single cells withina heterogeneous cell population. However, when the cell typeof interest is extremely rare, as most stem and progenitor cellsare, several sources of artifact must be addressed. The impor-tance of flow cytometry as a driving force for stem cellresearch was demonstrated in a focused issue of the journalexactly three years ago (1). This current focus issue of Cytome-try A is devoted to the topic of stem cells due to numerouscurrent innovations and discoveries.Applications of stem cells include several disciplines,from embryogenesis, adult tissue maintenance, and repair,and more recently, cancer as well as for toxicity screening anddisease modeling. All of these topics are represented in thisissue, with special emphasis on the role of analytic and pre-parative flow cytometry in the elucidation of stem cell pheno-type and function, and best laboratory practices as they applyto flow cytometry. Image and flow cytometry together withcell sorting have revolutionized the study of stem cell biologyand the implications of these cells and their progeny indevelopmental biology, tissue engineering, and cellulartherapy. The number of parameters and the speed of theirsimultaneous measurements in single cells has continued toincrease with advances in hardware, reagents, and analyticalsoftware (2).

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.

See related article, pages 648–656 The ability to generate induced pluripotent stem (iPS) cells from somatic cells by the overexpression of a limited number of stem cell-related genes has generated great excitement and interest in the biomedical research community including cardiovascular researchers. The pioneering study by Yamanaka and colleagues showing that overexpression of Oct3/4 , Sox2 , Klf4 , and c-Myc could reprogram mouse fibroblasts to a pluripotent state similar to that of embryonic stem (ES) cells opened major new avenues of research.1 This epigenetic reprogramming was rapidly extrapolated to the human system using either the same combination of reprogramming factors or a slightly different combination of transgenes ( OCT4 , NANOG , SOX2 , LIN28 ).2–4 Like embryonic stem (ES) cells, iPS cells can be used for basic developmental biology research and also as a cell source to generate theoretically unlimited quantities of desired cell types such as cardiomyocytes. Such differentiated cells types can be used in a wide range of basic research studies and potentially in clinical applications, which not only include cellular therapies but also drug discovery and safety testing. One appealing aspect of human iPS cells compared to human ES cells is that they can be more readily generated without specialized expertise and access to human embryos, which also avoids the ethical challenges associated with human embryo research. Potentially the most powerful advantage of iPS cells over ES cells is that they can be generated from any patient to produce genetically identical pluripotent cells that can create human disease models or generate patient-specific cells for therapy. Already a number of iPS cell human disease models have been generated,5,6 and proof-of-principle iPS cellular therapies have been pioneered in mouse models.7–9 Despite the speed at which the iPS cell field is racing forward, we …

Aging is a natural process defined as a progressive decline in physiological functions which lead to increased risk of diseases and death. Recent advances in antiaging intervention have focused on stem cell-based therapies and cell reprogramming. The development of stem cell reprogramming to fight the aging process has recently become important issue in antiaging strategies. Stem cell-based therapies and cell reprogramming have provided various strategies to alter somatic cell identity into induced-pluripotent stem cell. Stem cells are defined as pluripotent cells that possess both the abilities of self-renewal and differentiation toward numerous cell types. Cell reprogramming is simply composed of deleting cell memory and rewriting new identity of somatic cell. Stem cell reprogramming has provided enormous insight on regenerative medicine for antiaging. This chapter has focused on potential role of stem cell reprogramming to slow down aging process.

Reprogramming is one of the most essential areas of research in stem cell biology. Despite this importance, the mechanism and correlates of reprogramming remain largely unknown. In this study, we investigated the cytoplasmic remodeling and changes in metabolism that occur during reprogramming and differentiation of pluripotent stem cells. Specifically, we examined the cellular organelles of three pluripotent stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and epiblast stem cells (EpiSCs), by electron microscopy. We found that the cellular organelles of primed pluripotent EpiSCs were more similar to those of naive pluripotent ESCs and iPSCs than somatic cells. EpiSCs, as well as ESCs and iPSCs, contain large nuclei, poorly developed endoplasmic reticula, and underdeveloped cristae; however, their mitochondria were still mature relative to the mitochondria of ESCs and iPSCs. Next, we differentiated these pluripotent stem cells into neural stem cells (NSCs) in vitro and compared the morphology of organelles. We found that the morphology of organelles of NSCs differentiated from ESCs, iPSCs, and EpiSCs was indistinguishable from brain-derived NSCs. Finally, we examined the changes in energy metabolism that accompanied mitochondrial remodeling during reprogramming and differentiation. We found that the glycolytic activity of ESCs and iPSCs was greater compared with EpiSCs, and that the glycolytic activity of EpiSCs was greater compared with NSCs differentiated from ESCs, iPSCs, and EpiSCs. These results suggest that a change in the cellular state is accompanied by dynamic changes in the morphology of cytoplasmic organelles and corresponding changes in energy metabolism.