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.     

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.     

胚胎干细胞治疗心肌梗死的研究进展

胚胎干细胞 (ES细胞 )是一种多能细胞 ,来源于囊胚期胚胎 ,具有很强的自我更新能力 ,并能分化成很多细胞类型。体外 ,ES细胞能自发聚集形成胚胎体 (EB) ,分化成许多种细胞类型 ;ES细胞注射到免疫缺陷的小鼠体内 ,产生畸胎瘤 ,其中包含有三个胚层的细胞。添加生长因子或与其它细胞共培养等方法可以促进ES细胞体外分化为心肌细胞 ,筛选后移植到梗死的心肌 ,可以提高心脏功能 ,是治疗心肌梗死的一种很有潜力的方法     

Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

Dermal fibroblast-derived growth factors restore the ability of beta(1) integrin-deficient embryonal stem cells to differentiate into keratinocytes

Embryonal stem (ES) cells that are homozygous null for the beta(1) integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/beta(1)-null chimeric mice. The failure of beta(1)-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce beta(1)-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from beta(1)-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and beta(1)-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulated differentiation of keratin 14-positive beta(1)-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of beta(1) integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

人胚胎干细胞传代培养及细胞化学染色特性

目的 体外建立人胚胎干细胞传代培养方法,研究人胚胎干细胞细胞化学染色特性.方法 以小鼠胚胎成纤维细胞作为饲养层传代培养人胚胎干细胞,检测人胚胎干细胞、自发分化克隆及拟胚体的细胞化学染色特性.结果 人胚胎干细胞在小鼠胚胎成纤维细胞饲养层上传30代以上其形态保持不变;人胚胎十细胞碱性磷酸酶、过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性,自发分化克隆细胞阳性程度明显减弱;人胚胎干细胞形成的拟胚体碱性磷酸酶染色弱阳性,过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性.结论 小鼠胚胎成纤维细胞能支持人胚胎干细胞传代培养,细胞化学染色结果能初步鉴别人胚胎干细胞未分化特性.     

Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Self-renewal vs. differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.     

Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells

The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodies of different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport and metabolism of chemical and bioactive substances and prevent the damaging effects.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

Hedgehog signaling is required for the differentiation of ES cells into neurectoderm

Mouse embryonic stem cells can differentiate in vitro into cells of the nervous system, neurons and glia. This differentiation mimics stages observed in vivo, including the generation of primitive ectoderm and neurectoderm in embryoid body culture. We demonstrate here that embryonic stem cell lines mutant for components of the Hedgehog signaling cascade are deficient at generating neurectoderm-containing embryoid bodies. The embryoid bodies derived from mutant cells are also unable to respond to retinoic acid treatment by producing nestin-positive neural stem cells, a response observed in cultures of heterozygous cells, and contain cores apparently arrested at the primitive ectoderm stage. The mutant cultures are also deficient in their capacity to differentiate into mature neurons and glia. These data are consistent with a role for Hedgehog signaling in generating neurectoderm capable of producing the appropriate neuronal and glial progenitors in ES cell culture.     

Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Insulin-like growth factors promote vasculogenesis in embryonic stem cells

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.