STEM CELL THERAPY IN ACUTE RENAL FAILURE

描述(由申请人提供)： 目前急性肾功能衰竭的治疗选择有限。如果肾上皮细胞不能再生，肾功能衰竭的恢复是不完整的。由于干细胞具有不同的发育潜力，我们建议检验小鼠造血干细胞(HSCs)和小鼠胚胎干细胞(ES细胞)可以帮助急性肾功能衰竭后肾小管再生的假设。在我们的初步研究中，我们从雄性ROSA 26小鼠身上分离出HSC，并将它们注射到雌性C57BL小鼠体内，使其发生肾脏缺血/再灌注损伤。移植后4周，在女性受者肾小管中检测到Lac Z阳性细胞。男性特异性SRY基因的存在以及女性受者肾脏中Y染色体阳性信号的细胞证实了男性供者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X-Gal染色供体标记和肾小管转运蛋白抗体标记肾细胞来检查外源HSCs是否可以转分化为有功能的肾小管细胞。我们将测试注射不同数量的HSCs的剂量反应，并将检测HSCs对小鼠双侧肾缺血/再灌注损伤的影响。肾脏中供体来源细胞的数量将与血清尿素氮和肌酐水平相关。我们还将测试干细胞因子(SCF)和粒细胞集落刺激因子(G-CSF)动员内源性HSCs是否可以加速缺血损伤后肾功能的恢复。在第二个目标中，我们将检查是否可以诱导ES细胞分化为肾脏细胞。使用一种在肾小管和发育中的胃管上皮细胞中特异表达GFP的转基因小鼠株，我们将产生一种ES细胞系，这将使我们能够鉴定和分离活的肾小管上皮细胞。ES细胞将被诱导形成类胚体，然后在生长因子的作用下进一步培养，这是已知的诱导人类ES细胞表达肾脏细胞标志的条件。用FRCS分选法分离GFP阳性细胞。表达GFP的肾小管上皮细胞在缺血再灌注损伤后注入肾被膜下。肾脏切片将用GFP和肾小管转运蛋白的抗体进行染色，以测试移植的ES细胞来源的肾小管上皮细胞的功能作用。这一建议的结果将使人们能够对干细胞的可塑性和胚胎干细胞的肾小管发育有新的理解，并为用干细胞替代疗法治疗肾脏疾病，如急性肾功能衰竭开辟了可能性。

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.