STEM CELL THERAPY IN ACUTE RENAL FAILURE

描述（由申请人提供）： 目前急性肾衰竭的治疗选择有限。如果肾上皮细胞不能再生，则从肾衰竭的恢复是不完全的。由于干细胞具有不同的发育潜力，我们建议测试的假设，小鼠造血干细胞（HSC）和小鼠胚胎干细胞（ES细胞）可以帮助急性肾功能衰竭后的肾小管再生。在我们的初步研究中，我们从雄性Rosa 26小鼠中分离出HSC，并将其注射到患有肾缺血/再灌注损伤的雌性C57 BL小鼠中。在HSC移植后4周，我们在雌性受体肾的肾小管中检测到Lac Z阳性细胞。雄性特异性SRY基因的存在，以及雌性受者肾脏中Y染色体阳性信号的细胞证实了雄性供体HSC来源的细胞已定位于肾脏。在该提案的第一个目标中，我们将通过用X-gal染色肾组织作为供体标记物，并用肾小管转运蛋白抗体染色肾细胞标记物，来检查外源性HSC是否可以转分化为功能性肾小管细胞。我们将测试对注射不同数量的HSC的剂量反应，并将检查HSC在双侧肾缺血/再灌注损伤小鼠中的作用。肾脏中供体来源细胞的数量将与血清BUN和肌酐水平相关。我们还将测试是否动员内源性造血干细胞与干细胞因子（SCF）和G-CSF可以加速缺血性损伤后肾功能的恢复。在第二个目标中，我们将研究ES细胞是否可以被诱导分化为肾细胞。使用一种独特的转基因小鼠，表达GFP特异性在肾小管上皮细胞和发展中的GU道，我们将产生一个ES细胞系，使我们能够识别和分离肾小管上皮细胞。ES细胞将被诱导形成胚状体，然后进一步与生长因子一起培养，已知的条件是诱导人ES细胞表达肾细胞标志物。将使用FRCS分选分离GFP阳性细胞。在缺血/再灌注损伤后，将表达GFP的肾小管上皮细胞注射到肾包膜下。肾脏切片将用GFP和肾小管转运蛋白的抗体染色，以测试移植的ES细胞衍生的肾小管上皮细胞的功能作用。该提案的结果将允许对HSC的可塑性和ES细胞的肾小管发育进行新的理解，并开辟了用干细胞替代疗法治疗肾脏疾病（如急性肾衰竭）的可能性。

DESCRIPTION (provided by applicant): Current treatment options for acute renal failure are limited. The recovery from renal failure is incomplete if renal epithelial cells fail to regenerate. Since stem cells have diverse developmental potential, we propose to test the hypothesis that mouse hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ES cells) can aid in the regeneration of renal tubules after acute renal failure. In our preliminary studies, we isolated HSCs from male Rosa 26 mice and injected them into female C57 BL mice that had renal ischemia/reperfusion injury. At 4 weeks after HSC transplantation, we detected Lac Z positive cells in the renal tubules of female recipient kidneys. The presence of the male specific SRY gene, and the cells with Y chromosome positive signals in female recipient kidneys confirmed that male donor HSC-derived cells had located to the kidneys. In the first aim of the proposal, we will examine whether exogenous HSCs can transdifferentiate to functional renal tubular cells by staining the kidney tissues with X-gal for the donor marker, and with antibodies to tubular transporters for renal cell markers. We will test the dose response to various numbers of HSCs injected and will examine the effect of HSCs in mice with bilateral renal ischemia/reperfusion injury. The number of donor-derived cells in the kidneys will be correlated with serum BUN and creatinine levels. We will also test whether mobilization of endogenous HSCs with stem cell factor (SCF) and G-CSF can accelerate the recovery of renal function after ischemic injury. In the second aim, we will examine if ES cells can be induced to differentiate to renal cells. Using a unique strain of transgenic mice that express GFP specifically in the epithelial cells of renal tubules and the developing GU tract, we will generate an ES cell line that will allow us to identify and isolate live tubular epithelial cells. ES cells will be induced to form embryoid bodies, and then further cultured with growth factors, conditions known to induce human ES cells to express renal cell markers. GFP positive cells will be isolated with FRCS sorting. The GFP expressing tubular epithelial cells will be injected under the capsule of the kidneys after ischemia/reperfusion injury. The kidney sections will be stained with antibodies to GFP and tubular transporters to test the functional role of transplanted ES cell-derived tubular epithelial cells. The results of this proposal will permit novel understanding of plasticity of HSCs and renal tubular development from ES cells and open up the possibility of treating renal diseases, such as acute renal failure, with stem cell replacement therapy.