Epigenetic stability of embryonic stem cells and developmental potential

Recent studies highlight the tremendous potential of human embryonic stem (ES) cells and their derivatives as therapeutic tools for degenerative diseases. However, derivation and culture of ES cells can induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Research on human and mouse stem cells indicates that developmental, cancer-related genes, and genes regulated by genomic imprinting are particularly susceptible to changes in DNA methylation. Together with the occurrence of genetic alterations, epigenetic instability needs to be monitored when considering human stem cells for therapeutic and technological purposes. Here, we discuss the maintenance of epigenetic information in cultured stem cells and embryos and how this influences their developmental potential.     

Assessment of drug induced developmental toxicity using human embryonic stem cells

Embryonic stem (ES) cells are unique as they have the potential to be generated in large numbers and the ability to differentiate into the three germ layers via embryoid body (EB) formation. This property could be utilized as an index to study initial mammalian development. We have investigated the utility of a comprehensively characterized human ES (hES) cell line (ReliCellhES1) for testing the embryotoxic effects of compounds using cytotoxicity assays. Further, we performed real time gene expression analysis to check the alterations in germ layer markers expression upon drug treatment. The results show that assays using hES cells could serve as a reliable, sensitive and robust method to assess embryotoxic potential of compounds. They also provide a proof of concept that hES cells can be used as an in vitro model to demonstrate developmental toxicity, and to examine the germ layer-specific effects on differentiating EBs.     

人胚胎干细胞的研究

来自着床前的囊胚和早期人胚胎的人胚胎干细胞是未分化的多能干细胞,具有无限增殖和分化的潜力,这种特性使之在基础研究和移植治疗中具有广泛的应用。尤其是胚胎干细胞可以产生任何类型的可供临床使用的细胞、组织和器官的潜力,将会带来一场医学革命。     

US policies on human embryonic stem cells

The United States is a federal union with separate state jurisdictions. In part owing to the sometimes heated debate about public support for human embryonic stem-cell (ESC) research, there has been restricted federal support and little central regulation of this research to date. Instead, guidelines developed by scientific organizations have set principles for oversight and good practice for this research. These guidelines are functioning well, have influenced developing state regulations and, one hopes, will affect any future federal regulation.     

Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.     

An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo

Embryonic stem cells (ES) cells were injected into host blastocysts either in groups of 10-15 cells or as single cells in order to test their developmental potential in the developing embryo. The analysis of midgestation chimaeras, by electrophoretic separation of glucose phosphate isomerase (GPI) isozymes, showed that ES cells were capable of colonizing trophectoderm and primitive endoderm derivatives at a low frequency, as well as producing a high rate of chimaerism in tissues of the fetus and extraembryonic mesoderm.     

Therapeutic potential of human embryonic stem cells in type 2 diabetes mellitus

AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications were enrolled and injected with hE SC lines as per the defined protocol.The patients were assessed using Nutech functional score(NFS),a numeric scoring scale to evaluate the patients for 11 diagnostic parameters.Patients were evaluated at baseline and at the end of treatment period 1(T1).All the parameters were graded on the NFS scale from 1to 5.Highest possible grade(HPG)of 5 was considered as the grade of best improvement.RESULTS:Overall,94.8%of the patients showed improvement by at least one grade of NFS at the end of T1.For all the 11 parameters evaluated,54%of patients achieved HPG after treatment.The four essential parameters(improvement in glycated hemoglobin(HbA 1c)and insulin level,and fall in number of other oral hypoglycemic drugs with and without insulin)are presented in detail.For Hb A1c,72.6%of patients at the end of T1 met the World Health Organization cut off value,i.e.,6.5%of HbA 1c.For insulin level,65.9%of patients at the end of T1 were able to achieve HPG.After treatment,the improvement was seen in 16.3%of patients who required no more than two medications along with insulin.Similarly,21.5%of patients were improved as their dosage regimen for using oral drugs was reduced to 1-2 from 5.CONCLUSION:hE SC therapy is beneficial in patients with diabetes and helps in reducing their dependence on insulin and other medicines.     

Glycosphingolipids of human embryonic stem cells

The application of human stem cell technology offers theoretically a great potential to treat various human diseases. However, to achieve this goal a large number of scientific issues remain to be solved. Cell surface carbohydrate antigens are involved in a number of biomedical phenomena that are important in clinical applications of stem cells, such as cell differentiation and immune reactivity. Due to their cell surface localization, carbohydrate epitopes are ideally suited for characterization of human pluripotent stem cells. Amongst the most commonly used markers to identify human pluripotent stem cells are the globo-series glycosphingolipids SSEA-3 and SSEA-4. However, our knowledge regarding human pluripotent stem cell glycosphingolipid expression was until recently mainly based on immunological assays of intact cells due to the very limited amounts of cell material available. In recent years the knowledge regarding glycosphingolipids in human embryonic stem cells has been extended by biochemical studies, which is the focus of this review. In addition, the distribution of the human pluripotent stem cell glycosphingolipids in human tissues, and glycosphingolipid changes during human stem cell differentiation, are discussed.     

Requirements for human embryonic stem cells

‘Requirements for Human Embryonic Stem Cells’ is the first set of guidelines on human embryonic stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for human embryonic stem cells, which is applicable to the quality control for human embryonic stem cells. It was originally released by the China Society for Cell Biology on 26 February 2019 and was further revised on 30 April 2020. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human embryonic stem cells for applications.     

Immunogenicity of human embryonic stem cells

Human embryonic stem cells (HESC) are pluripotent stem cells isolated from the inner cell mass of human blastocysts. With the first successful culturing of HESC, a new era of regenerative medicine was born. HESC can differentiate into almost any cell type and, in the future, might replace solid organ transplantation and even be used to treat progressive degenerative diseases such as Parkinson’s disease. Although this sounds promising, certain obstacles remain with regard to their clinical use, such as culturing HESC under well-defined conditions without exposure to animal proteins, the risk of teratoma development and finally the avoidance of immune rejection. In this review, we discuss the immunological properties of HESC and various strategic solutions to circumvent immune rejection, such as stem cell banking, somatic cell nuclear transfer and the induction of tolerance by co-stimulation blockade and mixed chimerism.     

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.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.     

Murine embryonic stem cells as a model for human embryonic stem-cell research

Over the last several decades, murine embryonic stem cells (mESCs) have been used as a model for human embryonic stem cell (hESC) research. The relevance of this approach has not yet been proven. There is a great deal of evidence that is indicative of substantial differences between these two cell types. An analysis of the literature shows that the differences concern ESC proliferation, self-renewal, and differentiation. Consequently, mESC may be considered as a model object for hESC studies only for some aspects of their biology. The alternative model objects, such as primate ESC, are also discussed briefly in this review.     

Mouse embryonic stem cells exhibit indefinite proliferative potential

The proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life-span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of "crisis" or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ-line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.     

Derivation of human embryonic stem cells with NEMO deficiency

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent, have the capacity to differentiate into hematopoietic progenitors, and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease.