Transplantable neural progenitor populations derived from Rhesus monkey embryonic stem cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% +/- 8.1%)populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65% - 80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

星形胶质细胞存在L-型钙通道的新证据

下载PDF阅读器以纯化培养的小鼠星形胶质细胞(Astrocyte,AS)为实验材料,采用激光共聚焦钙成像和荧光分光光度计技术,探讨钙激动剂Bay k8644和钙拮抗剂nimodipine对星形胶质细胞胞内钙离子浓度的影响.结果显示,Bay k8644在0.0001、0.001和0.01 mmol/L浓度下均可显著增加星形胶质细胞的细胞内钙水平,而nimodipine在0.001、0.01和0.1 mmol/L浓度下均可显著降低星形胶质细胞胞内钙水平,并阻止KCl引起的细胞内钙升高.上述结果表明星形胶质细胞对L-型钙通道激动剂和拮抗剂的反应与神经元的反应相似,提示星形胶质细胞胞膜上也存在L-型钙通道.

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。

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.