Progress in studies on the induction and differentiation of embryonic stem cells

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocyst stage embryo, have the potential to give rise to an entire organism and to generate every body cell type. Much improvement has been made in the field of induction and differentiation of ESCs during the last two years, such as the ESCs differentiation into germ cells (2003) and the cloning of human ESCs (2004), both of which were chosen respectively as one of the top ten achievements evaluated by academic journals. Great attention was also paid to the research of the new genes which could maintain ESCs in the undifferentiated state and the research of the induction and differentiation of ESCs.     

Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

Directed neural differentiation of human embryonic stem cells (ESCs) enables researchers to generate diverse neuronal populations for human neural development study and cell replacement therapy. To realize this potential, it is critical to precisely understand the role of various endogenous and exogenous factors involved in neural differentiation. Cell density, one of the endogenous factors, is involved in the differentiation of human ESCs. Seeding cell density can result in variable terminal cell densities or localized cell densities (LCDs), giving rise to various outcomes of differentiation. Thus, understanding how LCD determines the differentiation potential of human ESCs is important. The aim of this study is to highlight the role of LCD in the differentiation of H9 human ESCs into neuroectoderm (NE), the primordium of the nervous system. We found the initially seeded cells form derived cells with variable LCDs and subsequently affect the NE differentiation. Using a newly established method for the quantitative examination of LCD, we demonstrated that in the presence of induction medium supplemented with or without SMAD signaling blockers, high LCD promotes the differentiation of NE. Moreover, SMAD signaling blockade promotes the differentiation of NE but not non-NE germ layers, which is dependent on high LCDs. Taken together, this study highlights the need to develop innovative strategies or techniques based on LCDs for generating neural progenies from human ESCs.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.     

Defining pluripotent stem cells through quantitative proteomic analysis

Embryonic stem cells (ESCs) are at the center stage of intense research, inspired by their potential to give rise to all cell types of the adult individual. This property makes ESCs suitable candidates for generating specialized cells to replace damaged tissue lost after injury or disease. However, such clinical applications require a detailed insight of the molecular mechanisms underlying the self-renewal, expansion and differentiation of stem cells. This has gained further relevance since the introduction of induced pluripotent stem cells (iPSCs), which are functionally very similar to ESCs. The key property that iPSCs can be derived from somatic cells lifts some of the major ethical issues related to the need for embryos to generate ESCs. Yet, this has only increased the need to define the similarity of iPSCs and ESCs at the molecular level, both before and after they are induced to differentiate. In this article, we describe the proteomic approaches that have been used to characterize ESCs with regard to self-renewal and differentiation, with an emphasis on signaling cascades and histone modifications. We take this as a lead to discuss how quantitative proteomics can be deployed to study reprogramming and iPSC identity. In addition, we discuss how emerging proteomic technologies can become a useful tool to monitor the (de)differentiation status of ESCs and iPSCs.     

干细胞命运决定的分子调控网络

来源于囊胚期胚胎内细胞团的胚胎干细胞具有独特的生物学特性,包括无限自我更新的能力以及分化为内胚层、中胚层和外胚层各种细胞的潜能.阐明胚胎干细胞全能性维持以及向各种特定细胞分化的分子机制,不仅有助于我们了解胚胎发育过程,而且将促进胚胎干细胞尽早应用于疾病治疗.本文主要就干细胞的一种命运决定过程,维持胚胎干细胞全能性或失去全能性开始分化,结合最新的研究进展讨论该过程中的分子调控网络,包括信号转导通路、表达调控网络以及表观遗传调控.     

Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this increased characterization of the cytoskeleton in pluripotent stem cells will allow for the better understanding and design of stem cell-based therapies.     

Germline potential of parthenogenetic haploid mouse embryonic stem cells

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.     

CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation

This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1A1-mediated metabolism of xenobiotics by cytochrome P450 on male germ cell differentiation by qRT-PCR and immunocytochemistry. Results showed that 17,742 DEGs were found during differentiation of ESCs into SSCs and enriched in 72 differently significant pathways. Thereinto, the metabolism of xenobiotics by cytochrome P450 was involved in the whole differentiation process of ESCs into SSCs and several candidate DEGs: CYP1A1, CYP3A4, CYP2D6, ALDH3B1, and ALDH1A3 were expressed with the same trend with RNA-Seq. Knockdown of CYP1A1 caused male germ cell differentiation under restrictions. Our findings showed that the metabolism of xenobiotics by cytochrome P450 was significantly different during the process of male germ cell differentiation and was persistently activated when we induced cESCs to differentiate into SSCs with RA in vitro, which illustrated that the metabolism of xenobiotics by cytochrome P450 played a crucial role in the differentiation process of ESCs into SSCs.     

Neural differentiation of embryonic stem cells is induced by signalling from non-neural niche cells.

BACKGROUND/AIMS: Embryonic stem cell (ESC) transplantation offers new therapeutic strategies for neurodegenerative diseases and injury. However, the mechanisms underlying integration and differentiation of engrafted ESCs are poorly understood. This study elucidates the influence of exogenous signals on ESC differentiation using in vitro modelling of non-stem/stem cell interactions. METHODS: Murine ESCs were co-cultured with endothelial cells and astrocytes or conditioned medium obtained from endothelial or astrocyte cultures. After 7 days of co-culture isolated RNA was analysed using RT-PCR for the expression of pluripotency marker oct-4, neural progenitor marker nestin, and neurofilament (NFL), an early marker of neuronal lineage commitment. The presence of the glial cell surface marker A2B5 was determined in ESCs by flow cytometry. RESULTS: Neuronal differentiation was inhibited in ESCs when grown in close vicinity to cerebral endothelial or glial cells. Under these conditions, ESC differentiation was predominantly directed towards a glial fate. However, treatment of ESCs with endothelial cell- or astrocyte-conditioned medium promoted neuronal as well as glial differentiation. CONCLUSION: Our results indicate that ESC fate is determined by endothelial and glial cells that comprise the environmental niche of these stem cells in vivo. The direction of differentiation processes appears to be dependent on humoral factors secreted by adjacent cell lines.     

Current focus of stem cell application in retinal repair

The relevance of retinal diseases, both in society’s economy and in the quality of people’s life who suffer with them, has made stem cell therapy an interesting topic forresearch. Embryonic stem cells(ESCs), induced pluripotent stem cells(i PSCs) and adipose derived mesenchymal stem cells(ADMSCs) are the focus in current endeavors as a source of different retinal cells, such as photoreceptors and retinal pigment epithelial cells. The aim is to apply them for cell replacement as an option for treating retinal diseases which so far are untreatable in their advanced stage. ESCs, despite the great potential for differentiation, have the dangerous risk of teratoma formation as well as ethical issues, which must be resolved before starting a clinical trial. i PSCs, like ESCs, are able to differentiate in to several types of retinal cells. However, the process to get them for personalized cell therapy has a high cost in terms of time and money. Researchers are working to resolve this since i PSCs seem to be a realistic option for treating retinal diseases. ADMSCs have the advantage that the procedures to obtain them are easier. Despite advancements in stem cell application, there are still several challenges that need to be overcome before transferring the research results to clinical application. This paper reviews recent research achievements of the applications of these three types of stem cells as well as clinical trials currently based on them.     

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-α (PDGFR-α) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-α and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.     

Wnt3a is involved in the early stage of miPSC and mESC haemopoietic differentiation

The Wnt/β-catenin signalling pathway is important in regulating not only self-renewal of haemopoietic progenitors and stem cells but also haemopoietic differentiation of ESCs (embryonic stem cells). However, it is still not clear how it affects haemopoietic differentiation. We have used a co-culture system for haemopoietic differentiation of mouse ESCs and iPSCs (induced pluripotent stem cells) in which the Wnt3a gene-modified OP9 cell line is used as stromal cells. The number of both Flk1+ and CD41+ cells generated from both co-cultured mouse ESCs and mouse iPSCs increased significantly, which suggest that Wnt3a is involved in the early stages of haemopoietic differentiation of mouse ESCs and mouse iPSCs in vitro.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.     

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.