Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Identification and characterization of a MBP isoform specific to hypothalamus in orange-spotted grouper (Epinephelus coioides)

Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role informing and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oliaodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution. (C) 2007 Elsevier B.V.. All rights reserved.

猕猴胚胎干细胞无饲养层、无血清培养及诱导分化成O2A 胶质前体细胞的研究

灵长类胚胎干细胞（ES 细胞）不仅能为研究生殖发育生物学基础理论提供良好的 模型，而且可为细胞替代治疗提供大量的供体细胞，因此具有重要的研究价值。当前 灵长类ES 细胞研究还有很多问题需要解决，如分离建立更多的胚胎干细胞系，优化培 养体系，提高ES 细胞定向分化为特定细胞的比例，研究ES 细胞自我更新和分化的机 制等。本文一方面概括了灵长类ES 细胞的研究进展，另一方面并对制备抗体，免疫外 科手术法分离灵长类胚胎内细胞团，建立猕猴ES 细胞的无饲养层、无血清培养体系和 诱导猕猴ES 细胞分化成高纯度的O2A 神经胶质前体细胞进行了研究。主要结论如下： 1）分别以猕猴脾脏淋巴细胞和人外周血单个核细胞作为免疫原，免疫日本大耳白兔， 得到免疫血清。在补体介导的细胞毒作用下，兔抗人和兔抗猕猴免疫血清可以裂解人 和猕猴囊胚滋养层细胞，从而分离出内细胞团，用于分离培养人和猕猴胚胎干细胞。2） 猕猴ES 细胞在以层粘连蛋白（laminin）为胞外基质，含转化生长因子beta1（TGFβ1） 的无血清培养基（SFM）中可以稳定的增殖至少22 代，保持不分化，并具有分化成三 个胚层细胞的能力。进一步的研究发现去除TGFβ1 后，猕猴ES 细胞出现分化，整合 素表达降低，推测TGFβ1 可能通过促进猕猴ES 细胞整合素的表达，加强其与胞外基 质的相互作用，从而维持ES 细胞的自我更新。然而猕猴ES 细胞不能在纤粘连蛋白 （fibronectin）和明胶上生长。3）无饲养层、无血清培养体系中长期培养的猕猴ES 细 胞，分化出拟胚体，14 天的拟胚体在血清中分化培养一周后，在含碱性成纤维生长因 子bFGF、表皮生长因子EGF 和胰岛素＋转铁蛋白＋亚硒酸钠ITS 的培养基中培养， 获得97％的O2A 胶质前体细胞，得到的O2A 细胞能够稳定增殖，并且可以自发分化 为II 型星型胶质细胞和少突胶质细胞。本实验的结果有助于猕猴ES 细胞分离建系和培 养系统的优化、推动猕猴ES 细胞自我更新和诱导为神经胶质细胞机制的研究，便于建 立ES 细胞替代治疗的猕猴模型，从而为人类ES 细胞的临床疾病治疗提供参考。