Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16 h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Chromosome number variation in three mouse embryonic stem cell lines during culture

Although mouse embryonic stem cell lines (mESCs) have been established since 1981, systematic studies about chromosomal changes during culture are lacking. In this study, we report the results of a cytogenetic analysis performed on three mESC lines (named UPV02, UPV06 and UPV08) cultured for a period of 3 months. At time intervals, the variation of the chromosome number together with the expression of markers of the undifferentiated status, i.e., OCT-4, SSEA-1, FOM-1 and alkaline phosphatase activity, were determined. The three mESC lines showed a progressive loss of euploid metaphases during the 3 months period of culture. Chromosome abnormalities were accumulated at the latest passages analysed. Metacentric chromosomes were the most frequent chromosome abnormality observed throughout the period of culture. Interestingly, in coincidence with, or few passages after, the drop of euploidy, the alkaline phosphatase activity was partially or totally lost, whereas the OCT-4, SSEA-1 and FOM-1 stem markers were always positive throughout the period of culture. Our results remark the necessity to perform the karyotype analysis during culture in order to develop new culture conditions to maintain the correct chromosome complement in long-term culture of mESC lines.     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14 days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation

Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.