A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.     

Maintenance of human pluripotent stem cells using 4SP-hFGF2-secreting STO cells

Human embryonic stem cells (hESCs) are typically cultured on fibroblast feeder cells or in fibroblast conditioned medium supplemented with fibroblast growth factor 2 (FGF2, also known as bFGF). FGF signaling appears to be important for hESC self-renewal and is required to enable the culture of hESCs in an undifferentiated state. In this study, we generated a transgenic fibroblast feeder line stably expressing a secretable FGF4 signal peptide tagged hFGF2 (4SP-hFGF2). The expression of this transgene functionally replaced the requirement for exogenous FGF2 when using these cells as feeders for the maintenance of hESCs. Under these conditions, hESCs maintained the typical marker of pluripotency assessed after long term culture, while still retaining the capacity for differentiation to all three germ layers. This transgene could be applied to mass produce 4SP-hFGF2 protein, serving to be an economical and effective strategy for culturing pluripotent stem cells as feeder cells.     

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.     

Basic fibroblast growth factor and its receptors in human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs), which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF) into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.     

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.     

Requirement of B-Raf,C-Raf,and A-Raf for the growth and survival of mouse embryonic stem cells

Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells.     

不同培养体系对绵羊类胚胎干细胞分离、传代的影响

以小鼠胚胎成纤维细胞(MEF)为饲养层, 研究了用Knockout血清替代品(Knockout serum replacement, KSR)代替胚胎干细胞(Embryonic stem cells, ES cell)培养液中的胎牛血清(FBS)和向含KSR的基础培养液中添加40%的小鼠ES细胞条件培养液(ES cell conditioned medium, ESCCM)对绵羊类ES细胞分离、克隆效率的影响。发现使用含FBS的基础培养液最多可以把绵羊类ES细胞传至3代, 而使用KSR和添加ESCCM能促进绵羊类ES细胞的分离和克隆, 所获得的类ES细胞分别可稳定传至第5和8代。同时对类ES细胞进行核型分析、AKP染色及体外分化能力检测, 证实所分离的类ES细胞符合ES细胞的主要特征。由此认为, 与FBS相比KSR更加适于绵羊类ES细胞的分离与培养; 而小鼠ES细胞在生长过程中可能分泌某些重要的细胞因子, 从而达到促进绵羊ES细胞增值的作用。     

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

Inhibition of Caspase-mediated Anoikis Is Critical for Basic Fibroblast Growth Factor-sustained Culture of Human Pluripotent Stem Cells

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells, we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture, which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture, although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand, all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells, our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation.     

Generation of trophoblast stem cells from rabbit embryonic stem cells with BMP4

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal.     

MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal

MEK/ERK signaling plays a crucial role in a diverse set of cellular functions including cell proliferation, differentiation and survival, and recently has been reported to negatively regulate mouse embryonic stem cell (mESC) self-renewal by antagonizing STAT3 activity. However, its role in human ESCs (hESCs) remains unclear. Here we investigated the functions of MEK/ERK in controlling hESC activity. We demonstrated that MEK/ERK kinases were targets of fibroblast growth factor (FGF) pathway in hESCs. Surprisingly, we found that, in contrast to mESCs, high basal MEK/ERK activity was required for maintaining hESCs in an undifferentiated state. Inhibition of MEK/ERK activity by specific MEK inhibitors PD98059 and U0126, or by RNA interference, rapidly caused the loss of self-renewal capacity. We also showed that MEK/ERK signaling cooperated with phosphoinositide 3-kinase (PI3K)/AKT signaling in maintaining hESC pluripotency. However, MEK/ERK signaling had little or no effect on regulating hESC proliferation and survival, in contrast to PI3K/AKT signaling. Taken together, these findings reveal the unique and crucial role of MEK/ERK signaling in the determination of hESC cell fate and expand our understanding of the molecular mechanisms behind the FGF pathway maintenance of hESC pluripotency. Importantly, these data make evident the striking differences in the control of self-renewal between hESCs and mESCs.     

Feeder layer- and serum-free culture of human embryonic stem cells

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.     

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.