Mesenchymal regulation of fetal intestinal development and adult regeneration

项目概要 炎症性肠病代表上皮修复不足和结直肠癌 (CRC) 美国癌症死亡的第二大原因，反映了肠道干细胞的破坏 （ISC）稳态。 ISC 部分依赖于平衡间质信号梯度 隐窝细胞（包括 ISC）的增殖与分化能力。此外，ISC 可以再生 通过隐窝细胞的去分化，在消融后有效地进行。目前尚不清楚哪种信号或间充质 细胞促进这种再生过程。我的初步研究发现了三种不同的间充质细胞 类型，其中两种协调 BMP 信号梯度。高 PDGFRA 表达细胞， 对应于最近描述的特细胞，最靠近上皮，在绒毛处最丰富 碱基，并表达高水平的 BMP 配体。低 PDGFRA 表达细胞包含两种 亚群：仅在隐窝下方发现的 CD81 片段，表达高水平的 BMP 拮抗剂 Gremlin1 和位于隐窝上方和周围但缺乏的 CD81- 部分（CP 细胞） 格雷姆1.在分离的隐窝上皮与这些严格纯化的细胞类型中的每一种的共培养中， CD81 PDGFRAlo 细胞在功能上取代了所有添加生长因子的培养基，而 CP 细胞也这样做 最低限度，而远程细胞则不然。此外，体内 Grem1 细胞的消融会导致 ISC 的损失， 因此我将这些细胞称为滋养细胞。这些不同的间充质细胞群如何调节 胎儿发育过程中的 ISC 可塑性和丢失过程中的 ISC 再生尚不清楚。我假设 远细胞、CP 和滋养细胞功能在发育过程中不断进化，导致稳定的隐窝细胞的形成。 绒毛生态位，并且一些相同的群体在 ISC 消融后促进隐窝再生。在 目标 1，我将研究特细胞、CP 细胞和 小鼠胎儿 ISC 发育过程中的滋养细胞。我将确定这些细胞何时出现 开发，研究其完整的转录谱，并使用已建立的隐窝共培养 方法来定义他们的支持角色。在目标 2 中，我将测试这些间充质细胞如何反应 减少 ISC 损失并促进隐窝再生。使用 ISC 再生的体内测定，结合 通过批量和单细胞 RNAseq 以及体内基因敲除，我将测试哪些细胞促进 ISC 再生，特别是如果这是通过超生理 BMP 抑制发生的。总的来说，这些 研究将为胎儿 ISC 的发育和调节提供基本见解 发育和再生，对肠道生物学和疾病具有广泛的影响。

Project Summary Inflammatory bowel disorders represent insufficient epithelial repair and colorectal cancer (CRC), the second leading cause of cancer deaths in the United States, reflects disruption of intestinal stem cell (ISCs) homeostasis. ISCs depend in part on underlying mesenchymal signaling gradients that balance proliferation versus differentiation capacity of crypt cells, including ISCs. Moreover, ISCs regenerate efficiently after ablation, by de-differentiation of crypt cells. It is unknown which signals or mesenchymal cells promote this regenerative process. My preliminary studies uncover three distinct mesenchymal cell types, two of which orchestrate BMP signaling gradients. High PDGFRA- expressing cells, which correspond to recently described telocytes, lie closest to the epithelium, are most abundant at the villus base, and express high levels of BMP ligands. Low PDGFRA-expressing cells contain two subpopulations: a CD81+ fraction found exclusively below crypts and expressing high levels of the BMP antagonist Gremlin1, and a CD81- fraction (CP cells) which resides above and around crypts but lacks Grem1. In co-cultures of isolated crypt epithelium with each of these strictly purified cell types, CD81+PDGFRAlo cells functionally replace all growth factor-supplemented media, while CP cells do so minimally and telocytes do not. Furthermore, ablation of Grem1+ cells in vivo results in loss of ISCs, hence I call these cells trophocytes. How each of these distinct mesenchymal cell populations regulates fetal ISC plasticity during development and ISC regeneration during loss, is unknown. I hypothesize that telocyte, CP, and trophocyte functions evolve during development, leading to genesis of a stable crypt- villus niche and that some of the same populations promote crypt regeneration after ISC ablation. In Aim 1, I will investigate the molecular profiles and functions of telocytes, CP cells, and trophocytes during mouse fetal ISC development. I will determine when these cells arise during development, investigate their full transcriptional profiles, and use an established crypt co-culture method to define their supportive roles. In Aim 2, I will test how these mesenchymal cells respond to ISC loss and promote crypt regeneration. Using in vivo assays of ISC regeneration, combined with bulk and single-cell RNAseq and in vivo genetic knockouts, I will test which cells promote ISC regeneration and specifically if this occurs through supraphysiologic BMP inhibition. Collectively, these studies will provide fundamental insights into the development and regulation of ISCs in fetal development and regeneration, with broad implications for intestinal biology and disease.