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.     

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.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E-ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ cells as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency.     

Autophagy in human embryonic stem cells

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.     

Derivation of an interaction/regulation network describing pluripotency in human

Identification of the key genes/proteins of pluripotency and their interrelationships is an important step in understanding the induction and maintenance of pluripotency. Experimental approaches have accumulated large amounts of interaction/regulation data in mouse. We investigate how far such information can be transferred to human, the species of maximum interest, for which experimental data are much more limited. To address this issue, we mapped an existing mouse pluripotency network (the PluriNetWork) to human. We transferred interaction and regulation links between genes/proteins from mouse to human on the basis of orthologous relationship of the genes/proteins (called interolog mapping). To reduce the number of false positives, we used four different methods: phylogenetic profiling, Gene Ontology semantic similarity, gene co-expression, and RNA interference (RNAi) data. The methods and the resulting networks were evaluated by a novel approach using the information about the genes known to be involved in pluripotency from the literature. The RNAi method proved best for filtering out unlikely interactions, so it was used to construct the final human pluripotency network. The RNAi data are based on human embryonic stem cells (hESCs) that are generally considered to be in a (primed) epiblast stem cell state. Therefore, we assume that the final human network may reflect the (primed) epiblast stem cell state more closely, while the mouse network reflects the (unprimed/naïve) embryonic stem cell state more closely.     

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.     

Human Embryonic Stem Cell Lines with Lesions in FOXP3 and NF1

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders. We obtained preimplantation genetic diagnosed (PGD) blastocyst staged embryos with genetic mutations that cause human disorders and derived hESCs from these embryos. We applied laser assisted micromanipulation to isolate the inner cell mass from the blastocysts and plated the ICM onto the mouse embryonic fibroblast cells. Two hESC lines with lesions in FOXP3 and NF1 were established. Both lines maintain a typical undifferentiated hESCs phenotype and present a normal karyotype. The two lines express a panel of pluripotency markers and have the potential to differentiate to the three germ layers in vitro and in vivo. The hESC lines with lesions in FOXP3 and NF1 are available for the scientific community and may serve as an important resource for research into these disease states.     

Loss of Pluripotency in Human Embryonic Stem Cells Directly Correlates with an Increase in Nuclear Zinc

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.