Nuclear reprogramming by human embryonic stem cells

Embryonic stem cells have two unique properties. They are capable of indefinite self-renewal and, being pluripotent, they can differentiate into all possible cell types, including germ cells. A new study by Cowan et al. (2005) published in Science shows that human embryonic stem cells are able to reprogram the nuclei of fully differentiated human somatic cells, apparently conferring on them a pluripotent state.     

体细胞重编程为多潜能干细胞的研究进展

人类的胚胎干细胞（embryonic stem cells,ES cells）可以用来治疗很多疾病,但是如果通过核移植来获得与供体或者患者相匹配的ES细胞,就会受到人卵母细胞来源等条件的制约。这就促使了将体细胞重编程为多潜能细胞这样一种技术策略的发展,其中包括将分化细胞与ES细胞融合,在卵细胞、ES细胞或多潜能癌细胞的抽提物中孵育,强制多潜能因子过表达等具体的方法。通过这些途径引出了一些核功能的重编程以及相应的DNA甲基化修饰、组蛋白翻译后修饰,使体细胞表达特定的多潜能因子,转变为类似胚胎干细胞的多潜能细胞。     

X-chromosome epigenetic reprogramming in pluripotent stem cells via noncoding genes

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally ‘naïve’, with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.     

Cellular reprogramming by single-cell fusion with mouse embryonic stem cells in pig

Fusion of differentiated somatic cells with pluripotent stem cells can be used for cellular reprogramming, but the efficiency to obtain hybrid cells is extremely low. Here, we explored a novel cell fusion system, termed single-cell fusion, the efficiency was significantly improved verified by fusion of mouse embryonic stem cells (mESCs), comparing to traditional polyethylene glycol fusion. Then, we employed the optimized system to perform cell fusion of porcine embryonic fibroblasts (PEFs) and porcine pluripotent stem cells (pPSCs) with mESCs. The hybrid cells showed both red and green fluorescence and expressed species-specific genes of mouse and pig to evidence that the fusion was successful. The hybrid cells displayed characteristics similar with mESCs, including colony morphology, alkaline phosphatase positive and formation of embryoid body, and the expressions of core pluripotent factors OCT4, NANOG, and SOX2 of the pig were induced in the mESC/PEF hybrid cells. The results indicate PEFs and pPSCs could be reprogrammed by mESCs via the single-cell fusion. Taking advantage of the hybrid cells to investigate the signaling pathways depended on the pluripotency of pig, we suggest the transforming growth factor-β signaling pathways may play important roles. In summary, the single-cell fusion is highly efficient, and we believe in the future it will be widely used in the application and fundamental research.     

Generating pluripotent stem cells: Differential epigenetic changes during cellular reprogramming

Pluripotent stem cells hold enomous potential for therapuetic applications in tissue replacement therapy. Reprogramming somatic cells from a patient donor to generate pluripotent stem cells involves both ethical concerns inherent in the use of embryonic and oocyte-derived stem cells, as well as issues of histocompatibility. Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)-derived by ectopic expression of four reprogramming factors in donor somatic cells-are superior in terms of ethical use, histocompatibility, and derivation method. However, iPSC also show genetic and epigenetic differences that limit their differentiation potential, functionality, safety, and potential clinical utility. Here, we discuss the unique characteristics of iPSC and approaches that are being taken to overcome these limitations.     

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Background

Reprogrammed cells, including induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cells (NT-ESCs), are similar in many respects to natural embryonic stem cells (ESCs). However, previous studies have demonstrated that iPSCs retain a gene expression signature that is unique from that of ESCs, including differences in microRNA (miRNA) expression, while NT-ESCs are more faithfully reprogrammed cells and have better developmental potential compared with iPSCs.

Results

We focused on miRNA expression and explored the difference between ESCs and reprogrammed cells, especially ESCs and NT-ESCs. We also compared the distinct expression patterns among iPSCs, NT-ESCs and NT-iPSCs. The results demonstrated that reprogrammed cells (iPSCs and NT-ESCs) have unique miRNA expression patterns compared with ESCs. The comparison of differently reprogrammed cells (NT-ESCs, NT-iPSCs and iPSCs) suggests that several miRNAs have key roles in the distinct developmental potential of reprogrammed cells.

Conclusions

Our data suggest that miRNAs play a part in the difference between ESCs and reprogrammed cells, as well as between MEFs and pluripotent cells. The variation of miRNA expression in reprogrammed cells derived using different reprogramming strategies suggests different characteristics induced by nuclear transfer and iPSC generation, as well as different developmental potential among NT-ESCs, iPSCs and NT-iPSCs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-488) contains supplementary material, which is available to authorized users.     

胚胎干细胞分化过程中的表观遗传调控

作为一类既有自我更新能力,并具有多向分化潜能的细胞,胚胎干细胞具有非常重要的理论研究意义和临床应用前景。近期以胚胎干细胞为模型,研究有关干细胞分化的表观遗传调控已成为新的研究热点。本文就胚胎干细胞分化过程中DNA甲基化、组蛋白修饰、非编码RNA调控以及与胚胎干细胞分化密切相关的表观遗传学动态变化做一概述,对表观遗传学改变与胚胎干细胞分化关系的基础研究进行探讨。     

TLR4 expression in mouse embryonic stem cells and in stem cell-derived vascular cells is regulated by epigenetic modifications

Embryonic stem (ES) cells and ES cell-derived differentiated cells can be used in tissue regeneration approaches. However, inflammation may pose a major hurdle. To define the inflammatory response of ES and ES cell-derived vascular cells, we exposed these cells to LPS. With the exception of MIF no significant cytokine mRNA levels were observed either at baseline or after stimulation. Further experiments revealed that these cells do not express TLR4. Analysis of the DNA methylation status of the TLR4 upstream region showed increased methylation. Moreover, in vitro methylation suppressed TLR4 promoter activity in reporter gene assays. ChIP assays showed that in this region histones H3 and H4 are hypoacetylated in ES cells. Interestingly, 5-aza-dC or TSA partially relieves this gene repression. Finally, the increased levels of TLR4 observed in ES cells after treatment with 5-aza-dC or TSA confer responsiveness to LPS, as induction of IL-6 and TNFalpha mRNA was detected in endotoxin stimulated ES cells.     

Induction of lung-like cells from mouse embryonic stem cells by decellularized lung matrix

Decellularization of tissues is a recently developed technique mostly used to provide a 3-dimensional matrix structure of the original organ, including decellularized lung tissues for lung transplantation. Based on the results of the present study, we propose new utilization of decellularized tissues as inducers of stem cell differentiation. Decellularized lung matrix (L-Mat) samples were prepared from mouse lungs by SDS treatment, then the effects of L-Mat on differentiation of ES cells into lung cells were investigated. ES cell derived-embryoid bodies (EBs) were transplanted into L-Mat samples and cultured for 2 weeks. At the end of the culture, expressions of lung cell-related markers, such as TTF-1 and SP-C (alveolar type II cells), AQP5 (alveolar type I cells), and CC10 (club cells), were detected in EB outgrowths in L-Mat, while those were not found in EB outgrowths attached to the dish. Our results demonstrated that L-Mat has an ability to induce differentiation of ES cells into lung-like cells.     

Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

Nucleostemin (NS) is a nucleolar GTP-binding protein that was first identified in neural stem cells, the functions of which remain poorly understood. Here, we report that NS is required for mouse embryogenesis to reach blastulation, maintenance of embryonic stem cell (ESC) self-renewal, and mammary epithelial cell (MEC) reprogramming to induced pluripotent stem (iPS) cells. Ectopic NS also cooperates with OCT4 and SOX2 to reprogram MECs and mouse embryonic fibroblasts to iPS cells. NS promotes ESC self-renewal by sustaining rapid transit through the G1 phase of the cell cycle. Depletion of NS in ESCs retards transit through G1 and induces gene expression changes and morphological differentiation through a mechanism that involves the MEK/ERK protein kinases and that is active only during a protracted G1. Suppression of cell cycle inhibitors mitigates these effects. Our results implicate NS in the maintenance of ESC self-renewal, demonstrate the importance of rapid transit through G1 for this process, and expand the known classes of reprogramming factors.