Differentiation of ES Cells into Renal Epithelia

描述（申请人提供）：急性肾衰竭是一种严重的临床问题，需要重症监护住院治疗和透析。急性肾衰的主要原因是近端小管急性小管坏死（ATN），这是由于毒物暴露或缺血后损伤引起的。本文对肾小管损伤的生理和分子生物学进行了较详细的研究。然而，尽管知识基础和透析的可用性不断增加，ATN后的死亡率并没有显著改善，仍保持在50%左右。患者从ATN中恢复能力的明显速率限制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存活的近端小管细胞保留和增殖，以便小管再生。因此，如果可以增加可用的近端小管祖细胞群，则可以显著增强ATN损伤小管的再生潜力。在本研究中，我们研究了胚胎干细胞是否可以分化为肾上皮细胞，以及胚胎干细胞是否可以整合到发育中的肾脏中。肾上皮来源于中胚层，在肾脏发育的早期阶段表达一种独特的基因组合。基于我们对早期肾脏发育的了解，我们将研究在中胚层发现的生长和分化因子的组合是否可以用来驱动胚胎干细胞沿着肾上皮谱系分化。胚胎干细胞将在不含LIF的情况下培养，并形成胚状体。这些胚胎样体将用类维生素a和生长因子联合处理，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，我们将分离胚胎干细胞衍生体，并将细胞注射到发育中的肾脏中。胚胎干细胞促进肾脏生长的能力可以通过各种组织学和免疫学方法进行检查。ES源性肾祖细胞帮助缓解ATN的能力将在动物模型中进行测试。由于胚胎干细胞可能代表着肾脏祖细胞的无限供应，这些实验将为基于细胞的治疗奠定基础，以提高ATN治疗的疗效。

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.