Direct conversion of fibroblasts to urothelial stem cells

项目总结 在泌尿外科手术中，尿路重建通常涉及利用小肠 更换泌尿系统的一段，结果是，患者可能会遭受连接的副作用 尿路和胃肠道。例如，接受尿流改道手术的患者面临风险。 由于这种做法，感染、电解质异常和肠梗阻的风险很高。如果一种更好的尿液替代品 肠道可用，尿流改道或涉及小肠的重建手术的结果将 会有很大的改进。这项提议的主要目标是开发一种来源自体的尿路上皮干细胞。 这可能被用于开发替代的膀胱或尿路上皮替代品。在这 我们假设尿路上皮干细胞可以通过成纤维细胞的直接转化而产生，并且可以 重建小鼠膀胱尿路上皮。首先，我们的目标是通过直接培养产生尿路上皮干细胞 转化，或转分化(目标1)，我们将通过过度表达转录因子来实现这一点 与膀胱发育和筛查基础以上和基础尿路上皮标志物有关。我们会 用有机化合物和多层集合体的功能分析来验证我们的筛选结果。 其次，我们将把尿路上皮干细胞分化过程中发生的表观遗传学变化映射到 超基底细胞(目标2)，我们将通过对对照膀胱器质进行OMNI ATAC-SEQ来实现这一点 和尿路上皮干细胞在基础和分化介质条件下的细胞器。通过确定以下方面的差异 染色质超可及区，我们将能够识别丰富的转录因子结合基序 干细胞和分化细胞状态。最后，我们将开发一种膀胱尿路上皮干细胞移植方案。 使用小鼠尿路上皮消融和损伤模型(目标3)。我们将确定尿路上皮干细胞 移植可以利用从以下来源获得的尿路上皮干细胞重建尿路上皮内的所有细胞类型 小鼠膀胱以及通过转分化获得的膀胱，我们将测试功能结果。如果 我们在这些目标上是成功的，我们将证明自体尿路上皮干细胞可以通过 直接转化成纤维细胞，我们将为膀胱替代组织工程建立细胞来源 也是以细胞为基础治疗通常通过外科手术治疗的尿路上皮疾病的基础 使用胃肠道重建，如严重放射性膀胱炎或严重间质性膀胱炎。这个 这项工作对人类健康的影响将是巨大的，因为这项工作可能会使尿流改道和 对于患有严重尿路上皮疾病的患者来说，重建手术是一种病态较少的手术选择.

PROJECT SUMMARY Reconstruction of the urinary tract in urologic surgery oftentimes involves utilizing the small intestine to replace a segment of the urinary system, and as a result, patients may suffer from the side effects of connecting the urinary and gastrointestinal tracts. Patients that undergo urinary diversion surgery, for example, are at risk of infection, electrolyte abnormalities, and ileus as a result of this practice. If a better substitute for the urinary tract were available, outcomes from urinary diversion or reconstructive surgery involving the small intestine would be drastically improved. The main goal of this proposal is to develop a source of autologous urothelial stem cells that can potentially be used towards the development of alternative bladder or urothelial substitutes. In this proposal we hypothesize that urothelial stem cells can be generated via direct conversion of fibroblasts and can reconstitute the bladder urothelium in the mouse. First, we will aim to generate urothelial stem cells via direct conversion, or transdifferentiation (Aim 1), and we will achieve this by overexpressing transcription factors associated with bladder development and screening for suprabasal and basal urothelial markers. We will validate our screening results with functional assays with organoids as well as multilayered assembloids. Second, we will map the epigenetic changes that take place during urothelial stem cell differentiation to suprabasal cells (Aim 2), and we will accomplish this by performing Omni ATAC-seq on control bladder organoids and urothelial stem cell organoids in basal and differentiation media conditions. By identifying differences in areas of chromatin hyperaccessibility, we will be able to identify transcription factor binding motifs enriched in stem cell and differentiated cell states. Finally, we will develop a bladder urothelial stem cell transplant protocol using mouse models of urothelial ablation and injury (Aim 3). We will determine if urothelial stem cell transplantation can reconstitute all cell types within the urothelium utilizing urothelial stem cells obtained from mouse bladders as well as those obtained from transdifferentiation, and we will test for functional outcomes. If we are successful in these aims, we will demonstrate that autologous urothelial stem cells can be generated via direct conversion of fibroblasts, and we will establish a source of cells for bladder substitute tissue engineering as well as a basis for cell-based therapy for disorders of the urothelium that are typically treated with surgical reconstruction using the gastrointestinal tract, such as severe radiation cystitis or severe interstitial cystitis. The impact of this work on human health will be significant as this work will potentially make urinary diversion and reconstruction surgery a much less morbid surgical option for patients with severe urothelial disorders.