Embryonic stem cells: a promising tool for cell replacement therapy

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.     

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formarion and their subsequent differentiation in a single three dimensional environment.     

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.     

Human embryonic stem cells: emerging technologies and practical applications

Human embryonic stem cells possess the unique ability to differentiate into any adult cell type. Recent advances in the understanding of stem cell biology make new applications possible for stem cell based technology. Of note, it is now possible to reprogram terminally differentiated human somatic cells into pluripotent cells that are functionally equivalent to embryonic stem cells. These induced pluripotent cells may become the substrate for future disease models and cell-based therapies. In addition, novel techniques for genetic manipulation have increased the ease with which genes can be modified into stem cells. In this review, we describe these novel technologies as well as developments in the understanding of basic biology of stem cell pluripotency and differentiation.     

Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。     

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.     

Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro

Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so-called 'embryoid bodies'. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.     

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     

Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.     

Embryonic stem cells

Embryonic stem cells have huge potential in the field of tissue engineering and regenerative medicine as they hold the capacity to produce every type of cell and tissue in the body. In theory, the treatment of human disease could be revolutionized by the ability to generate any cell, tissue, or even organ, 'on demand' in the laboratory. This work reviews the history of murine and human ES cell lines, including practical and ethical aspects of ES cell isolation from pre-implantation embryos, maintenance of undifferentiated ES cell lines in the cell culture environment, and differentiation of ES cells in vitro and in vivo into mature somatic cell types. Finally, we discuss advances towards the clinical application of ES cell technology, and some of the obstacles which must be overcome before large scale clinical trials can be considered.     

Enhancement by growth factors of cardiac myocyte differentiation from embryonic stem cells: a promising foundation for cardiac regeneration

Cell transplantation is a promising, still novel, potentially therapeutic approach for the treatment of heart diseases. Clinical applications require generation of large number of donor cells. Embryonic stem (ES) cells are capable of self-renewal apparently in an unlimited fashion, in vitro. Theoretically, they can differentiate into any cell type required for cell transplantation, including cardiac myocytes. Diverse growth factors have been implicated in programming diverse cellular processes, including development of the embryonic heart, ES cell self-renewal, and cardiac myocyte differentiation from ES cells. This review addresses the current understanding of the role of growth factors in the differentiation of cardiac myocytes from ES-embryoid body cell systems in vitro as well as cardiac regeneration in vivo.     

Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment.     

胚胎干细胞定向分化为心肌细胞研究进展

胚胎干细胞在体外可分化为 3个胚层的所有组织细胞。诱导人类胚胎干细胞定向分化为心肌细胞可为心肌梗死、心肌坏死等重大心脏疾病患者实施细胞治疗 ,也可作为种子细胞 ,用于构建供器官移植用的人造心脏 ;进一步可研究心肌细胞发育分化的分子机理及更直观的用于体外筛选人类心血管药物等。对人类胚胎干细胞及其定向分化为心肌细胞分子机理的研究进展及其所面临的问题作一综述。     

In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells

Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.     

Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia

Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 μm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 μm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.