Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency

The factors and signaling pathways controlling pluripotent human cell properties, both embryonic and induced, have not been fully investigated. Failure to account for functional heterogeneity within human embryonic stem cell (hESC) cultures has led to inconclusive results in previous work examining extrinsic influences governing hESC fate (self renewal vs. differentiation vs. death). Here, we attempt to reconcile these inconsistencies with recent reports demonstrating that an autologously produced in vitro niche regulates hESCs. Moreover, we focus on the reciprocal paracrine signals within the in vitro hESC niche allowing for the maintenance and/or expansion of the hESC colony-initiating cell (CIC). Based on this, it is clear that separation of hESC-CICs, apart from their differentiated derivatives, will be essential in future studies involving their molecular regulation. Understanding how extrinsic factors control hESC self-renewal and differentiation will allow us to culture and differentiate these pluripotent cells with higher efficiency. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine.     

R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal

Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/β-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.     

A molecular basis for human embryonic stem cell pluripotency

Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFβ, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFβ signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.     

Modulating glypican4 suppresses tumorigenicity of embryonic stem cells while preserving self-renewal and pluripotency

Self-renewal and differentiation of stem cell depend on a dynamic interplay of cell-extrinsic and -intrinsic regulators. However, how stem cells perceive the right amount of signal and at the right time to undergo a precise developmental program remains poorly understood. The cell surface proteins Glypicans act as gatekeepers of environmental signals to modulate their perception by target cells. Here, we show that one of these, Glypican4 (Gpc4), is specifically required to maintain the self-renewal potential of mouse embryonic stem cells (ESCs) and to fine tune cell lineage commitment. Notably, Gpc4-mutant ESCs contribute to all embryonic cell lineages when injected in blastocyts but lose their intrinsic tumorigenic properties after implantation into nude mice. Therefore, our molecular and functional studies reveal that Gpc4 maintains distinct stemness features. Moreover, we provide evidence that self-renewal and lineage commitment of different stem cell types is fine tuned by Gpc4 activity by showing that Gpc4 is required for the maintenance of adult neural stem cell fate in vivo. Mechanistically, Gpc4 regulates self-renewal of ESCs by modulating Wnt/β-catenin signaling activities. Thus, our findings establish that Gpc4 acts at the interface of extrinsic and intrinsic signal regulation to fine tune stem cell fate. Moreover, the ability to uncouple pluripotent stem cell differentiation from tumorigenic potential makes Gpc4 as a promising target for cell-based regenerative therapies. Stem Cells2012;30:1863-1874.     

Hydrogels as artificial matrices for human embryonic stem cell self-renewal

Human embryonic stem cells (hESCs) have the potential to differentiate into all cell types in the body and hold great promise for regenerative medicine; however, large-scale expansion of undifferentiated hESCs remains a major challenge. Self-renewal of hESCs requires culturing these cells on either mouse or human fibroblast cells (i.e., a feeder layer of cells), or on artificial extracellular matrices (ECMs) while supplementing the media with soluble growth factors. Here we report a completely synthetic ECM system composed of a semi-interpenetrating polymer network (sIPN), a polymer hydrogel, which was designed to allow the independent manipulation of cell adhesion ligand presentation and matrix stiffness. In the short term, hESCs that were cultured on the sIPN adhered to the surface, remained viable, maintained the morphology, and expressed the markers of undifferentiated hESCs. This was the first demonstration that a completely synthetic ECM can support short-term self-renewal of hESCs.     

The regulation of self-renewal in human embryonic stem cells

Human embryonic stem (hES) cells have the ability to self-renew while maintaining their pluripotency. The ability of stem cells to self-renew expansively is essential in both development and maintenance of adult tissues. ES cell lines were first generated from mouse blastocysts, these lines provided much needed information regarding ES cell propagation, growth factor dependence, and marker expression. However, the application potential of murine models is restricted in value because many differences between mouse and human ES cells have since been identified. The process of hES cells self-renewal appears to be regulated by many different pathways; however, the molecular mechanisms enabling this process are not fully characterized. Further defining these mechanisms will enable growth of hES cells under defined conditions and aid controlled differentiation of cells into specified lineages, in turn providing cells suitable for therapeutic applications. This review provides a summary of the mechanisms known to control self-renewal and pluripotency in hES cells.     

Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency

Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos; they can be cultured indefinitely and differentiated into many cell types in vitro. These cells therefore have the ability to provide insights into human disease and provide a potential unlimited supply of cells for cell-based therapy. Little is known about the factors that are important for maintaining undifferentiated hESCs in vitro, however. As a tool to investigate these factors, transfected hES clonal cell lines were generated; these lines are able to express the enhanced green fluorescent protein (EGFP) reporter gene under control of the OCT4 promoter. OCT4 is an important marker of the undifferentiated state and a central regulator of pluripotency in ES cells. These OCT4-EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells. Expression of OCT4-EGFP is colocalized with endogenous OCT4, as well as the hESC surface antigens SSEA4 and Tra-1-60. In addition, the expression is retained in culture for an extensive period of time. Differentiation of these cells toward the neural lineage and targeted knockdown of endogenous OCT4 expression by RNA interference downregulated the EGFP expression in these cell lines, and this correlates closely with the reduction of endogenous OCT4 expression. Therefore, these cell lines provide an easy and noninvasive method to monitor expression of OCT4 in hESCs, and they will be invaluable for studying not only OCT4 function in hESC self-renewal and differentiation but also the factors required for maintenance of undifferentiated hESCs in culture.     

Screening for genes essential for mouse embryonic stem cell self-renewal using a subtractive RNA interference library

The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.     

Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR?1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel?-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR?1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel?) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.     

FGF2 signaling in mouse embryonic fibroblasts is crucial for self-renewal of embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained undifferentiated (pluripotent) or differentiated to basically all functional cell types, depending on the culture conditions used. Culture of hESCs in the presence of medium conditioned by mouse embryonic fibroblasts (MEFs) can be used to keep hESCs undifferentiated. This observation suggests that MEFs produce factors required for the pluripotency of hESCs. The data presented here show that fibroblast growth factor 2 (FGF2) treatment of MEFs is crucial for the production of these factors. To identify the potential factors that are expressed in the presence of FGF2 in MEFs, a global expression profile analysis using microarrays was performed. This analysis indicated that 17 secreted factors are downregulated in the absence of FGF2. These factors include several ligands for known signaling receptors, extracellular proteases and components of the extracellular matrix, that may all be involved in signaling events. Surprisingly, we found that selective blocking of extracellular signal-regulated kinase (ERK) signaling by the MAPK/ERK kinase (MEK) inhibitor U0126 affected the expression of only some of the FGF2-regulated genes, suggesting FGF2-induced pathways that are independent of ERK signaling. It has been shown recently that activation of Activin/Nodal signaling and inhibition of bone morphogenetic protein signaling are required for the maintenance of pluripotency. Accordingly, among the 17 FGF2-regulated genes we found inhibin beta B that can lead to the assembly of Activin B and gremlin 1 that codes for an antagonist of bone morphogenetic proteins. This study identifies potentially important factors involved in the maintenance of pluripotency in hESCs and may allow the development of defined culture conditions without contaminating material from animal cells.     

Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency

Polycomb repressive complex 2 (PRC2) methylates histone H3 tails at lysine 27 and is essential for embryonic development. The three core components of PRC2, Eed, Ezh2, and Suz12, are also highly expressed in embryonic stem (ES) cells, where they are postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low- and high-passage Eed(null) ES cells to determine whether PRC2 is required for the maintenance of pluripotency. We report here that although developmental regulators are overexpressed in Eed(null) ES cells, both low- and high-passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors. Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells. We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency. Disclosure of potential conflicts of interest is found at the end of this article.     

Basic fibroblast growth factor support of human embryonic stem cell self-renewal

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.