Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 10⁵ hESCs are needed to form a 16 cm³ teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.     

Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status,Pluripotency, Cell Viability and Growth

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin^TM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin^TM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin^TM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin^TM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.     

Epigenetic Regulation of miR-302 by JMJD1C Inhibits Neural Differentiation of Human Embryonic Stem Cells

It has been recently reported that the regulatory circuitry formed by OCT4, miR-302, and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C, a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs, directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression, decreased BMP signaling, and enhanced TGFβ signaling. JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown, but not the control, cells within 3 days, accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together, our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.     

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.     

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche^1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment⁴ such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures². These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded⁵. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions² to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes¹. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.     

从人胚胎干细胞畸胎瘤中有效获取间充质干细胞和神经前体细胞

通过人胚胎干细胞(human embryonic stem cells,hESC)体外分化方法和畸胎瘤形成可以分化获得多种成体细胞．但目前尚不清楚是否可以从hESCs畸胎瘤中分离某些特异性细胞．通过体外筛选方法,有效地从hESCs畸胎瘤中分离出神经前体细胞(neural progenitor cells,NPCs)和间充质干细胞(mesenchymal stem cells,MSCs)．这种hESCs畸胎瘤来源的NPCs和MSCs与体内神经前体细胞和间充质干细胞有着相似的分子标记和特性,并具有进一步的分化潜能——分别可以诱导成为神经元、神经胶质细胞、脂肪细胞和骨骼细胞等．根据人胚胎干细胞畸胎瘤中含有不同分化阶段的外胚层、中胚层和内胚层的组织或细胞,认为人胚胎干细胞畸胎瘤可以作为另一个细胞来源以获取多种(包括人胚胎干细胞体外分化难以得到的)各种前体/干细胞和终末分化细胞．     

High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.     

Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.     

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.     

Embryonic Stem Cells: Spontaneous and Directed Differentiation

The specific structural features of embryonic stem cells and embryoid bodies and mechanisms of their differentiation in different cell types are considered. The mouse embryonic stem cells (line R1) formed multilayer colonies which enlarged as a result of fast cell division. Embryoid bodies that derived from embryonic stem cells consisted of an outer layer, an inner layer, and an internal cavity. The structure of cells of the outer and inner layers markedly differed. Spontaneous and directed differentiation of embryoid bodies is determined by some unspecific and specific factors (growth and differentiation factors and extracellular matrix proteins). Retinoic acid, the most commonly used inducer of differentiation of the embryonic stem cells, induces different types of differentiation when applied at different concentrations. The sequence of expression of tissue specific genes and proteins during differentiation of the embryonic stem cells in vitrois similar to that in vivo.     

Vitronectin promotes oligodendrocyte differentiation during neurogenesis of human embryonic stem cells

We demonstrate enhanced differentiation of oligodendrocytes during neurogenesis of human embryonic stem cells (hESCs) using an extracellular matrix protein, vitronectin (VN). We show that VN is expressed in the ventral part of the developing human spinal cord. Combined treatment of retinoic acid, sonic hedgehog, and noggin in the presence of VN allows hESCs to differentiate into O4-positive oligodendrocytes. Particularly, VN profoundly promotes the derivation of oligodendrocyte progenitors that proliferate and differentiate into oligodendrocytes in response to mitogenic and survival factors. These results support the beneficial effect of VN on oligodendrocytic differentiation of hESCs.     

SETD7 Regulates the Differentiation of Human Embryonic Stem Cells

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.