Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21?d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E(2) as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.     

Heparan Sulfate Is Required for Embryonic Stem Cells to Exit from Self-renewal

Pluripotent embryonic stem cells (ESCs) must select between alternative fates of self-renewal and lineage commitment at each division during continuous proliferation. Heparan sulfate (HS) is a highly sulfated polysaccharide and is present abundantly on the ESC surface. In this study, we investigated the role of HS in ESC self-renewal by examining Ext1^−/− ESCs that are deficient in HS. We found that Ext1^−/− ESCs retained their self-renewal potential but failed to transit from self-renewal to differentiation upon removal of leukemia inhibitory factor. Furthermore, we found that the aberrant cell fate commitment is caused by defects in fibroblast growth factor signaling, which directly retained high expression of the pluripotency gene Nanog in Ext1^−/− ESCs. Therefore, our studies identified and defined HS as a novel factor that controls ESC fate commitment and also delineates that HS facilitates fibroblast growth factor signaling, which, in turn, inhibits Nanog expression and commits ESCs to lineage differentiation.     

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.     

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 μm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.     

Short-term immunosuppression promotes engraftment of embryonic and induced pluripotent stem cells

Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation.     

Elimination of tumorigenic stem cells from differentiated progeny and selection of definitive endoderm reveals a Pdx1+ foregut endoderm stem cell lineage

Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.     

Loss of discordant cells during micro-mass differentiation of embryonic stem cells into the chondrocyte lineage

Embryonic stem cells (ESCs) have attracted particular interest in regenerative medicine because of their unlimited self-renewal and multipotentiality for differentiation. Spontaneous differentiated ESCs display heterogeneous multipotent cell populations and generate teratomas in vivo, with process by which ESCs differentiate into specific lineages remaining unclear. In this study, we focused on the in vitro chondrocyte differentiation of ESCs through micro-mass without using an embryoid body (EB) step and observed the unique characteristics of cartilage formation coupled with endochondral ossification in vivo. This approach resulted in an aggressive loss of discordant cells by apoptosis, which was accompanied by significant changes in gene expression during the course of ESC differentiation into chondrocytes. Unlike EB formation where discordant cells remain trapped within aggregates, micro-mass permits cells to die, leave the group and/or form a new group in response to changes in gene expression. Our observations suggest that the cell death that accompanies ESC micro-mass differentiation helps purify a terminally differentiated cell population and selects for targeted end points within a suitable microenvironment.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types?for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.     

Knockdown of p53 suppresses Nanog expression in embryonic stem cells

Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.     

APA微囊微环境影响胚胎干细胞增殖分化的体外研究

以小鼠胚胎T细胞（embryonic stem cell,ESC）为模型,在牛理条件F对ESC进行微囊化包封、培养,并利用免疫组织化学技术及RT-PCR方法检测其生长及未分化状态,以期建立微囊化ESC这一体外培养模型,同时明确海藻酸钠-聚赖氨酸-海藻酸钠（alginate-poly-lysine-alginate,APA）微囊微环境对ESC增殖及分化潜能的影响。结果表明：ESC能够在微囊（包括液化型及非液化型）或微球（海藻酸钙胶珠）内生长良好,但因生长环境存在差异,其表现的生长行为各具特征。比较其它类型,ESC在液化型APA微囊内的存活期限最长。经体外维持培养3周以上,仍能持续表达胚胎源未分化T细胞的标志性蛋白AP,SSEA-1及转录因子Oct-4。为进一步明确微囊内增殖的ESC是台仍具有多向分化的干细胞潜能,应用机械破囊法释放微囊内ESC团,并在体外进行定向诱导。经过近3周的条件诱导,其结果为：细胞团DTZ染色阳性：anti-insulin免疫荧光检测阳性;且特异性表达Pdx-1,Ins-1基因。上述结果证明：APA微囊为ESC维持未分化状态的增殖提供了特殊的微环境,APA微囊内所形成的ESC团仍具有多向分化的干细胞潜能。     

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.     

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2–5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.     

Pluripotency genes overexpressed in primate embryonic stem cells are localized on homologues of human chromosomes 16, 17, 19, and X

While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes overexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiated progeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicated in neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions.