BRL条件培养基在ES细胞培养中的应用方法探讨

目的：探讨布法罗大鼠肝细胞条件培养基（Buffalo rat liver cell conditioned medium,BRL）在ES细胞培养中的应用方法。方法：ES细胞复苏后分别培养在BRL条件培养基、小鼠胚胎成纤维细胞饲养层（mouse enbryonic fibroblast,MEF）及合并应用BRL条件培养基和MEF饲养层的环境中,通过细胞计数、拟胚体计数和ES细胞集落边缘细胞分化状态比较ES细胞在三种培养基中生长和分化差异。结果：与BRL组比较,MEF组和BRL＋MEF组细胞生长较快（P＜0.01）,ES细胞集落边缘分化细胞较少;MEF组和BRL＋MEF组无明显差异。结论：在复苏后早期阶段ES细胞培养中,不宜单独应用BRL条件培养基,须用MEF饲养层或合并应用BRL条件培养基和MEF饲养层。     

In vitro differentiation of mouse embryonic stem cells into inner ear hair cell-like cells using stromal cell conditioned medium

Hearing loss is mainly caused by loss of sensory hair cells (HCs) in the organ of Corti or cochlea. Although embryonic stem (ES) cells are a promising source for cell therapy, little is known about the efficient generation of HC-like cells from ES cells. In the present study, we developed a single-medium culture method for growing embryoid bodies (EBs), in which conditioned medium (CM) from cultures of ST2 stromal cells (ST2-CM) was used for 14-day cultures of 4-day EBs. At the end of the 14-day cultures, up to 20% of the cells in EB outgrowths expressed HC-related markers, including Math1 (also known as Atoh1), myosin6, myosin7a, calretinin, α9AchR and Brn3c (also known as Pou4f3), and also showed formation of stereocilia-like structures. Further, we found that these cells were incorporated into the developing inner ear after transplantation into chick embryos. The present inner ear HC induction method using ST2-CM (HIST2 method) is quite simple and highly efficient to obtain ES-derived HC-like cells with a relatively short cultivation time.     

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.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac‐specific genes, including α‐MHC and MLC‐2V, was significantly decreased in EphB4‐null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac‐specific genes were significantly enhanced by co‐culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4‐deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes. J. Cell. Biochem. 111: 29–39, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.     

人胚胎干细胞培养系统的研究进展

人胚胎干细胞（hESC）具有永久的自我更新和多潜能分化能力,可在一定条件下定向分化为三个胚层的各种细胞。这些特性使其在再生医学（细胞治疗）、药物筛选及早期胚胎发育研究中具有重要的应用前景;但人胚胎干细胞培养系统中大量的动物源性物质和复杂的未知成份大大阻碍了其医学应用价值,所以建立一个没有动物源物质、成份确定的人胚胎干细胞培养系统足非常重要的。本文简要介绍了为适应hESC临床应用和基础研究的需要,改良其培养系统的研究进展。     

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.     

以支持细胞为饲养层培养小鼠精原干细胞

为探索精原干细胞(Spermatogonialstemcells,SSCs)体外自增殖的条件以及SSCs体外快速扩增的方法,以6-8日龄昆明乳鼠为材料,分离小鼠睾丸细胞,采用Percoll梯度离心法富集SSCs;以经丝裂霉素C处理的Sertoli细胞作饲养层,以DMEM为基本培养基,加入5%胎牛血清和103u/ml的白血病抑制因子(Leukemiainhibitoryfactor,LIF),体外培养SSCs;运用免疫荧光技术,以SSCs特异性表面分子Thy1为标志,对原代培养20d和传代培养14d的细胞进行鉴定。该培养体系下,SSCs贴壁时间为6h-9h,48h后可见细胞分裂,迅速增殖出现在接种12d以后。接种后第20d形成数十至上百个细胞的细胞团,细胞总数比接种时增加了45-245倍,100倍显微镜下观察可见,单位视野内细胞团数为26±4个。传代后细胞增殖较快。原代培养20d和传代培养14d的细胞均为Thy1阳性;而传代20d后,细胞周缘不整,有伪足出现,呈现出死亡迹象。该培养条比较适合SSCs短期快速增殖。     

Multicolor karyotype analyses of mouse embryonic stem cells

Summary The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointment due to inadequate ES cell sources. Both authors contributed equally to this work.     

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.     

Effects of thymic polypeptides on the thymopoiesis of mouse embryonic stem cells

The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.     

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.     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.     

An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.     

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long-term proliferation of human embryonic stem (hES) cells is currently achieved by co-culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re-plated on six different densities of proliferative mEFs. hES colonies cultured on 1 x 10(5) - 5 x 10(5) proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self-renewal.