Differentiation of ES Cells into Renal Epithelia

描述(申请人提供)：急性肾功能衰竭是一个严重的临床问题，需要重症监护、住院和透析。急性肾功能衰竭的主要原因是近端小管的急性肾小管坏死(ATN)，这是由于毒物暴露或缺血造成的损伤。对肾小管损伤的生理学和分子生物学进行了较为详细的研究。然而，尽管不断增加的知识基础和透析的可用性，ATN后的死亡率并没有显著改善，仍然保持在50%左右。ATN患者康复能力的明显限速步骤是近端小管的再生。再生可能取决于是否有足够的未受损或有活力的近端小管细胞存活和增殖，从而使小管重新出现。因此，如果可以增加可用的近端小管祖细胞数量，ATN受损小管的再生潜力将显著增强。在这项建议中，我们研究胚胎干细胞(ES)是否可以分化为肾上皮细胞，以及这种ES细胞是否可以整合到发育中的肾脏中。肾上皮细胞来源于中间中胚层，在肾脏发育的早期阶段表达独特的基因组合。基于我们对早期肾脏发育的了解，我们将研究在中间中胚层中发现的生长和分化因子的组合是否可以沿着肾上皮谱系驱动ES细胞分化。ES细胞将在没有LIF的情况下培养，并允许形成类胚体。这些拟胚体将用维甲酸和生长因子的组合处理，并分析肾上皮特异性基因的表达。一旦我们能够激活适当的基因电池，我们将分离ES细胞衍生的小体，并将这些细胞注入发育中的肾脏。然后，可以通过各种组织学和免疫学方法来检测ES细胞对肾脏生长的贡献。胚胎干细胞来源的肾祖细胞帮助缓解ATN的能力随后将在动物模型中进行测试。由于ES细胞可能代表着无限的肾祖细胞供应，这些实验将为基于细胞的治疗奠定基础，以提高ATN的治疗效果。

DESCRIPTION (provided by applicant): Acute renal failure is a serious clinical problem requiring intensive care hospitalization and dialysis. The leading cause of acute renal failure is acute tubular necrosis (ATN) of the proximal tubule due to injury after either toxicant exposure or ischemia. The physiology and molecular biology of tubular injury have been studied in detail. Yet despite this increasing knowledge base and the availability of dialysis, mortality following ATN has not improved significantly and remains around 50%. The apparent rate limiting step in the ability of patients to recover from ATN is the regeneration of the proximal tubule. Regeneration may depend on whether enough undamaged or viable proximal tubule cells remain and proliferate so that repopulation of the tubule can occur. Thus, if the available proximal tubule progenitor population could be increased, the potential for regeneration of ATN damaged tubules could be significantly enhanced. In this proposal, we examine whether embryonic stem (ES) cells can be differentiated into renal epithelial cells and whether such ES cells can be integrated into a developing kidney. The renal epithelia is derived from intermediate mesoderm and express a unique combination of genes during the early stages of kidney development. Based on our knowledge of early renal development, we will examine whether combinations of growth and differentiation factors found in the intermediate mesoderm can be used to drive ES cell differentiation along the renal epithelial lineage. ES cells will be cultured free of LIF and allowed to form embryoid bodies. These embryoid bodies will be treated with combinations of retinoids and growth factors and analyzed for the expression of renal epithelial specific genes. Once we are able to activate the appropriate battery of genes, we will dissociate the ES cell derived bodies and inject the cells into a developing kidney. The ability of ES cells to contribute to the growing kidney can then be examined by a variety of histological and immunological methods. The ability of ES derived renal progenitor cells to help alleviate ATN will then be tested in animal models. Since ES cells potentially represent an unlimited supply of renal progenitors, these experiments will lay the foundation for cell based therapies to improve the efficacy of ATN treatment.