Direct conversion of fibroblasts to urothelial stem cells

项目概要 泌尿外科手术中的尿路重建通常涉及利用小肠 替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面临风险 这种做法会导致感染、电解质异常和肠梗阻。如果有更好的替代尿 如果有可用的尿道，尿流改道或涉及小肠的重建手术的结果将 得到大幅度改善。该提案的主要目标是开发自体尿路上皮干细胞的来源 这有可能用于开发替代性膀胱或尿路上皮替代品。在这个 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，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目标 与膀胱发育和基底上和基底尿路上皮标志物筛查相关。我们会 通过类器官和多层组合体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 基底上细胞（目标 2），我们将通过对对照膀胱类器官进行 Omni ATAC-seq 来实现这一目标 以及基础和分化培养基条件下的尿路上皮干细胞类器官。通过识别差异 染色质超易接近的区域，我们将能够识别富含的转录因子结合基序 干细胞和分化细胞状态。最后，我们将制定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是否 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，我们将建立膀胱替代组织工程的细胞来源 以及通常通过手术治疗的尿路上皮疾病的细胞疗法的基础 使用胃肠道重建，例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，因为这项工作可能会导致尿流改道和 对于患有严重尿路上皮疾病的患者来说，重建手术是一种发病率低得多的手术选择。

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.