Direct conversion of fibroblasts to urothelial stem cells

成纤维细胞直接转化为尿路上皮干细胞

• 批准号: 10661523
• 负责人: Kris Prado
• 金额: $ 16.71万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661523
• 关键词: ATAC-seq; Ablation; Anatomy; Area; Autologous; Binding; Biological Assay; Bladder; Bladder Control; Bladder Injury; Bladder Urothelial Cell; Bladder Urothelium; Bypass; Cell Differentiation process; Cell Therapy; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclophosphamide; Cystitis; Data; Development; Disease; Electrolytes; Engineering; Epigenetic Process; Epithelium; Fibroblasts; Future; Gastrointestinal tract structure; Genetic; Genitourinary system; Goals; Growth; Health; Hemorrhage; Human; Ileus; Immunocompromised Host; Immunosuppression; Injury; Interstitial Cystitis; Intestines; Maps; Mesenchymal Stem Cells; Metabolic; Methods; Molecular; Morphology; Mus; Operative Surgical Procedures; Organoids; Outcome; Patients; Perioperative; Play; Process; Protocols documentation; Public Health; Reconstructive Surgical Procedures; Regenerative Medicine; Regulation; Reporter; Review Literature; Risk; Screening Result; Small Intestines; Sorting; Source; Stem cell transplant; Surgical complication; Teratoma; Testing; Tissue Engineering; Tissues; Transplantation; Transposase; Trauma; UPK2 gene; UPK3 gene; Urinary Diversion; Urinary system; Urinary tract; Urinary tract infection; Urologic Surgical Procedures; Urothelial Cell; Urothelium; Work; cell type; design; differentiation protocol; embryonic stem cell; functional outcomes; improved; in vivo; induced pluripotent stem cell; infection risk; intestinal epithelium; knock-down; mouse model; novel; overexpression; postoperative recovery; radiation cystitis; reconstitution; reconstruction; regenerative; screening; side effect; stem cell differentiation; stem cells; transcription factor; transdifferentiation; urinary; urologic; urothelial injury

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.

项目摘要 泌尿道手术中尿路的重建通常涉及利用小肠 更换尿液系统的一部分，结果，患者可能患有连接的副作用 尿和胃肠道。例如，接受尿液转移手术的患者处于危险之中 由于这种做法，感染，电解质异常和回肠。如果更好地替代尿液 可用区域，尿液转移或涉及小肠的重建手术的结果 大大改善。该提案的主要目标是开发自体尿路干细胞的来源 可以潜在地用于开发替代膀胱或尿路上皮替代物。在这个 建议我们假设可以通过成纤维细胞直接转化尿路上皮干细胞，并且可以 重建小鼠中的膀胱尿铺上皮。首先，我们将通过直接产生尿路上皮干细胞 转换或跨分化（AIM 1），我们将通过过表达转录因子来实现这一目标 与膀胱发育和筛查上紫外线和基底尿路上皮标记有关。我们将 通过使用类器官以及多层组合的功能测定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期间发生的表观遗传变化 胸骨上细胞（AIM 2），我们将通过对对照膀胱器官进行Omni Atac-Seq来实现这一目标 在基础和分化培养基条件下的尿路上皮干细胞器官。通过确定差异 染色质超敏度的区域，我们将能够识别富含的转录因子结合基序 干细胞和分化细胞态。最后，我们将开发膀胱尿路干细胞移植方案 使用尿路上皮消融和损伤的小鼠模型（AIM 3）。我们将确定尿路上皮干细胞是否 移植可以利用从尿路上皮中重建所有细胞类型，利用从 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 我们在这些目标方面取得了成功，我们将证明可以通过 成纤维细胞的直接转化，我们将建立膀胱替代组织工程的细胞来源 以及基于细胞的尿路上皮疾病基于细胞的疗法，这些治疗通常由外科手术治疗 使用胃肠道的重建，例如严重的膀胱炎或严重的间质性膀胱炎。这 这项工作对人类健康的影响将非常重要，因为这项工作可能会导致尿液转移和 重建手术对于严重尿路上皮疾病患者的病态手术选择要少得多。