FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation

Here, we show that as human embryonic stem cells (ESCs) exit the pluripotent state, NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here, we find that FGF2, acting through the MEK-ERK pathway, switches BMP4-induced human ESC differentiation outcome to mesendoderm, characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation, that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm, and that knockdown of NANOG greatly reduces T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway.     

Niche-mediated control of human embryonic stem cell self-renewal and differentiation

Complexity in the spatial organization of human embryonic stem cell (hESC) cultures creates heterogeneous microenvironments (niches) that influence hESC fate. This study demonstrates that the rate and trajectory of hESC differentiation can be controlled by engineering hESC niche properties. Niche size and composition regulate the balance between differentiation-inducing and -inhibiting factors. Mechanistically, a niche size-dependent spatial gradient of Smad1 signaling is generated as a result of antagonistic interactions between hESCs and hESC-derived extra-embryonic endoderm (ExE). These interactions are mediated by the localized secretion of bone morphogenetic protein-2 (BMP2) by ExE and its antagonist, growth differentiation factor-3 (GDF3) by hESCs. Micropatterning of hESCs treated with small interfering (si) RNA against GDF3, BMP2 and Smad1, as well treatments with a Rho-associated kinase (ROCK) inhibitor demonstrate that independent control of Smad1 activation can rescue the colony size-dependent differentiation of hESCs. Our results illustrate, for the first time, a role for Smad1 in the integration of spatial information and in the niche-size-dependent control of hESC self-renewal and differentiation.     

BMP4 initiates human embryonic stem cell differentiation to trophoblast

The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.     

Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis

Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies.     

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.     

Mechanisms that mediate stem cell self-renewal and differentiation

Stem cells have two common properties: the capacity for self-renewal and the potential to differentiate into one or more specialized cell types. In general, stem cells can be divided into two broad categories: adult (somatic) stem cells and embryonic stem cells. Recent evidence suggested that tumors may contain "cancer stem cells" with indefinite potential for self-renewal. In this review, we will focus on the molecular mechanisms regulating embryonic stem cell self-renewal and differentiation, and discuss how these mechanisms may be relevant in cancer cells.     

Wnt信号在胚胎干细胞自我更新和分化中的作用

胚胎干细胞分离自胚泡内细胞团,具有无限自我更新和多向分化潜能,有很大的医学应用前景。Wnt家族是一类分泌型的细胞信号传导蛋白,可以通过复杂的信号传递通路调控胚胎的早期发育,对细胞的分化、增殖及生长具有重要的调节作用。该文就Wnt信号通路调节胚胎干细胞的自我更新和分化作一综述。     

KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

Background

Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function.

Results

Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes.

Conclusions

Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation.     

Indole derivatives sustain embryonic stem cell self-renewal in long-term culture

Embryonic stem cells (ESCs), which have characteristics such as self-renewal, indefinite proliferation, and pluripotency, are thought to hold great promise for regenerative medicine. ESCs are generally cultured on mouse embryonic fibroblast (MEF) or MEF conditioned medium (MEF-CM). However, for therapeutic applications, it is preferable for ESCs to be cultured under chemically defined conditions. Here, we report synthetic compounds that allow expansion of undifferentiated mouse ESCs in the absence of MEF, Leukemia Inhibitory Factor (LIF), and Fetal Bovine Serum (FBS). ESCs cultured for more than 30 d in a serum-free medium supplemented with indole derivertives retained their characteristic morphology and expressed markers such as SSEA-1, OCT3/4, Rex-1, Sox2, and Nanog. They consistently differentiated into many types of cells, including neurons, muscle cells, and hepatocytes. These results indicate that our compounds provide a more efficient and safer large-scale culture system for pluripotent ESCs, and hence might contribute to the use of ESCs in therapeutic applications.