Expression of estrogen receptor-alpha and -beta, glucocorticoid receptor, and progesterone receptor genes in human embryonic stem cells and embryoid bodies

Human embryonic stem cells (hESCs) have the potential to differentiate into various cell types, and the three germ layers in vivo and in vitro. They are therefore useful in transplantation and tissue engineering. Here, we describe the expression patterns of selected steroid receptor mRNAs - estrogen receptor-alpha (ER-alpha), ER-beta, glucocorticoid receptor (GR), and progesterone receptor (PR) - in undifferentiated hESCs and embryoid bodies (EBs) cultured for 2, 4, and 6 d, as assessed by real-time PCR, in order to define the possible influence of steroid hormones on the differentiation of hESCs. These receptor mRNAs were expressed in undifferentiated hESCs and EBs. The expression of PR mRNA only decreased during the differentiation of EBs but not of hESCs. Immunohistochemical analysis gave strong staining of ER-alpha, ER-beta, and GR proteins in the nuclei of hESCs and EBs, whereas PR was not detected. We also examined the potential of these steroid hormones to direct the differentiation of hESCs in vitro. The expression of 11 cell-specific markers representing 3 germ layers and 5 tissue types was used to assess the differentiation of hESCs. We found that certain endodermal marker genes were either only expressed in the estrogen-treated group or their expression was stimulated in that group, suggesting that steroid hormones can control the differentiation of hESCs into various cell types.     

Coupled global and targeted proteomics of human embryonic stem cells during induced differentiation

Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.     

A versatile tool for tracking the differentiation of human embryonic stem cells

The ability of human embryonic stem cells (hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are powerful tools for tracking cell types of interest in differentiated cell populations.Here,we describc the construction of modular lentivectors containing different tissue-specific promoters(Tαl of α-tubulin:αP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla green fluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to bc traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.     

Loss of stem cell regenerative capacity within aged niches

This work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. Specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human embryonic stem cells (hESCs) and post-natal muscle stem cells (satellite cells). Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches. Significantly, this work establishes that hESC-derived factors enhance the regenerative potential of both young and, importantly, aged muscle stem cells in vitro and in vivo; thus, suggesting that the regenerative outcome of stem cell-based replacement therapies will be determined by a balance between negative influences of aged tissues on transplanted cells and positive effects of embryonic cells on the endogenous regenerative capacity. Comprehensively, this work points toward novel venues for in situ restoration of tissue repair in the old and identifies critical determinants of successful cell-replacement therapies for aged degenerating organs.     

Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs.     

FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells

Insulin-producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic β cells, but its role in human β cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation-related genes and improved glucose-stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856-treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature β cells from hESCs and clinical applications in regenerative medicine.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

FGF signalling inhibits neural induction in human embryonic stem cells

Human embryonic stem cells (hESCs) can exit the self-renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self-renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non-neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.     

Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10microM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM alpha-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.     

Spa-1 regulates the maintenance and differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent, whereby they can proliferate endlessly and differentiate into many different cell types. At the molecular level, little is known of the mechanisms underlying their capability for self-renewal and differentiation. In the present study, we established two new hESC lines (AMC-hES1 and AMC-hES2) and demonstrated the existence of a regulator that may be a key molecule in hESC dynamics. Spa-1 is a principal Ras-proximate 1 (Rap1) GTPase-activating protein in hematopoietic progenitor cells that regulates Rap1-related signal transduction and is expressed restrictively in human adult tissues (bone marrow, thymus, and spleen). To investigate its functions in hESCs, we examined spa-1 expression profiles during hESC differentiation and used RNA interference (RNAi) to downregulate spa-1 in these cells. Our results show that Spa-1 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation. In addition, the process of passing from the mode of self-renewal to that of differentiation in hESCs was regulated by spa-1 via Rap1/Raf/mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling. An RNAi expression vector against spa-1 (pSUPER.retro.puro) was transfected into hESCs, which were seen to differentiate into three germ layers in spite of being in the undifferentiated condition. Based on our findings, therefore, it appears that spa-1 may be involved in hESC dynamics, and our results provide fundamental information regarding the self-renewal and differentiation of hESCs.     

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase ζ (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.Human embryonic stem cells (hESCs)¹ are stem cells derived from the blastocyst inner cell mass. They are pluripotent; thus they are able to differentiate into any human cell type. The self-renewal capacity and pluripotency make hESCs an ideal system to study the processes of cell development and differentiation. Moreover hESC research is highly relevant for regenerative medicine, which aims at replacing or restoring tissue damaged by disease or injury through transplantation of functional hESCs (1,2). However, factors responsible for maintaining the undifferentiated and pluripotent nature of hESCs are still largely unknown. Before hESCs can be used for transplantation into the human body, reliable and reproducible protocols for differentiating them into specific cell types are needed. To create such protocols we need to develop a thorough understanding of the mechanisms maintaining the undifferentiated pluripotent nature of hESCs and those guiding their differentiation into specific lineages.A number of factors involved in the maintenance of pluripotency have been described over the last few years (3). It has also been demonstrated that overexpression of some of these factors in somatic cells is sufficient to turn them into pluripotent stem cells very similar to hESCs (4–8). However, it is apparent that the processes occurring during such transformation are extremely complex. A large number of factors and pathways are involved in maintaining the pluripotent state and regulating self-renewal and differentiation. The process of specific hESC differentiation into distinct cell types is even less understood. Most current attempts to directionally differentiate hESCs are based on sequential application of empirically selected growth factors and consequent selection for markers expressed in the target cell types (9). A more systematic approach is needed to improve our understanding of the pathways that control the conversion of precursors into specific cell types, progressing toward the goal of reproducing these processes in vitro for the generation of functional cells and tissues for transplantation.Comprehensive quantitative analysis of the hESC proteome would mean an important advance in understanding the nature of “stemness,” pluripotency, and differentiation. Several studies targeting various aspects of the hESC proteome have already been reported (for reviews, see Refs. 10 and 11). The task, however, is so enormous that further detailed analysis and novel strategies are necessary and will be of high interest and importance. In this regard, MS-based quantitative proteomics and in particular stable isotope labeling by amino acids in cell culture (SILAC) may greatly facilitate the process of defining the mechanisms of hESC self-renewal and differentiation. With SILAC, the entire proteome of a given cell population is metabolically labeled by heavy, non-radioactive isotopic variants of amino acids, thus making it distinguishable by MS analysis (12). Thereafter two or more distinctly SILAC-labeled cell populations can be mixed and analyzed in one MS experiment that allows accurate quantitation of proteins from the different cellular states (13). This versatile strategy has been demonstrated to be very useful for comprehensive characterization of complex biological phenomena (14–21) including in-depth comparison of signaling pathways to identify control points determining cell fate of adult mesenchymal stem cells (22).Here we report a procedure for complete SILAC labeling of human ES cells. We show that these SILAC-encoded hESCs have preserved self-renewing undifferentiated status as well as pluripotent capabilities based on analysis of known markers. In addition, we further compared the overall proteomes and phosphoproteomes of SILAC-labeled hESCs and equivalent cells grown under conventional culture conditions. We next compared the membrane proteomes of undifferentiated and differentiated hESCs in a quantitative manner. Our analysis identified 811 membrane proteins, which to our knowledge is the largest data set of ES cell membrane proteome. This study also revealed 23 membrane proteins with large changes in their expression levels during the differentiation. Six of those cell surface molecules displayed more than 3-fold higher levels in the self-renewing cells, whereas the remaining 17 were identified as more abundant in the differentiated population. These may be useful as specific hESC markers for the corresponding ES cell state and help to shed light on the mechanisms for self-renewal and differentiation.     

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.     

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.