Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.     

Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells

Embryonic stem cells (ESCs) can self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives.Despite the importance of ESCs in developmental biology and their potential impact on regenerative medicine,the molecular mechanisms controlling ESC behavior are incompletely understood.Previously,activation of the canonical Wnt signaling pathway has been shown to contribute to mouse ESC self-renewal.Here we report that ectopic expression of Lef1,a component of the Wnt signaling pathway,has a positive effect on the self-renewal of mouse ESCs.Lef1 up-regulates Oct4 promoter activity and physically interacts with Nanog,two key components of the ESC pluripotency machinery.Moreover,siRNA for Lef1 induced mouse ESC differentiation.Our results thus suggest that in response to Wnt signaling Lef1 binds to stabilized β-catenin and helps maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.     

Cdk1 is required for the self-renewal of mouse embryonic stem cells

Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells. Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES cells.     

Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but clinical application of ES cells has been hindered by the lack of reliable selective differentiation methods. Differentiation into any lineage is partly dependent on the regulatory mechanisms of normal early development. Although several signals, including bone morphogenetic protein (BMP), Wnt and FGF, are involved in heart development, scarce evidence is available about the exact signals that mediate cardiomyocyte differentiation. While investigating the involvement of BMP signaling in early heart formation in the mouse, we found that the BMP antagonist Noggin is transiently but strongly expressed in the heart-forming region during gastrulation and acts at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by inhibition of BMP signaling.     

Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells

Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Signaling pathways regulating proliferation of mouse embryonic stem cells

Mouse embryonic stem (MES) cells possess joint abilities for unlimited proliferation and maintenance of pluripotency during long-term cultivation. The regulation of the cell cycle of these cells is of great interest. This review is focused on the regulation of the cell cycle of these cells via different signaling pathways (LIF-STAT3, PI3K-Akt, Wnt-β-catenin). The mechanisms underlying the unlimited proliferation of MES cells and their inability to long-term block of proliferation in response to DNA-damaging and stress factors are discussed. The functioning of negative (cyclin-kinase inhibitors and Rb) and positive (cyclin-kinase complexes and E2F factors) cell cycle regulators are also the topics of this survey. Permanent mitogenic stimuli are thought to prevent the induction of apoptosis; in any case, agents which cause a prolonged halt to proliferation without stimulating the onset of differentiation or the induction of apoptosis are currently unknown. Special concern is given to the role of the Wnt signaling pathway in sustaining the pluripotent state of MES cells. Cell cycle regulation by downstream targets of LIF-STAT3, PI3-kinase and Wnt-β-catenin pathways is discussed in light of the cooperative action of these pathways in the maintenance of undifferentiated states of MES cells.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells

ABSTRACT

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3β with IC₅₀ values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.     

Albumin-associated lipids regulate human embryonic stem cell self-renewal

Background

Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal.

Methodology/Principal Findings

In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect.

Conclusions/Significance

Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.     

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.     

Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.