Direct conversion of fibroblasts to urothelial stem cells

项目摘要 泌尿外科手术中的尿路重建通常涉及利用小肠， 更换泌尿系统的一部分，因此，患者可能会遭受连接的副作用。 泌尿道和胃肠道例如，接受尿流改道手术的患者 感染，电解质异常，肠梗阻，由于这种做法。如果有一种更好的尿替代品 如果有足够的肠道可用，尿流改道或涉及小肠的重建手术的结果将 得到大幅改善。这项提案的主要目标是开发一种自体尿路上皮干细胞来源 其可以潜在地用于开发替代性膀胱或尿路上皮替代物。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产生， 重建小鼠的膀胱尿道。首先，我们的目标是通过直接诱导产生尿路上皮干细胞， 转化或转分化（Aim 1），我们将通过过表达转录因子来实现这一点。 与膀胱发育和基底上和基底尿路上皮标志物的筛查相关。我们将 用类器官以及多层类胶质细胞的功能测定来验证我们的筛选结果。 其次，我们将绘制尿路上皮干细胞分化过程中发生的表观遗传变化， 我们将通过在对照膀胱类器官上进行Omni ATAC-seq来实现这一点 和基础和分化培养基条件下的尿路上皮干细胞类器官。通过识别 染色质超可及性区域，我们将能够识别富含 干细胞和分化细胞状态。最后，我们将制定一个膀胱尿路上皮干细胞移植方案 使用尿路上皮消融和损伤的小鼠模型（目的3）。我们将确定尿路上皮干细胞 移植可以利用从人尿道上皮获得的尿道上皮干细胞重建尿道上皮内的所有细胞类型。 小鼠膀胱以及从转分化获得的膀胱，我们将测试功能结果。如果 如果我们在这些目标上取得了成功，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，为膀胱替代组织工程建立细胞来源 也是基于细胞的治疗泌尿系统疾病的基础， 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。的 这项工作对人类健康的影响将是重大的，因为这项工作将有可能使尿流改道， 重建手术是严重尿路上皮疾病患者的一种较少病态的手术选择。

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.