Investigation and Translation of the Intestinal Stem Cell Niche

项目摘要/摘要 肠道被赋予了持续再生其上皮层的非凡能力，这两种能力都是为了维持 表达LGr5的活性和损伤/储备肠干介导的组织动态平衡和损伤修复 细胞(ISCs)和复杂的细胞和体液生态位。破坏这些再生特性会导致 吸收不良、感染性、炎症性和肿瘤性吸收障碍或屏障功能障碍或明显的过度生长 肠道疾病。因此，更好地了解ISCs及其利基环境对临床至关重要。 翻译这一生物学是美国国立卫生研究院肠道干细胞联盟(ISCC)的使命。作为当前的ISCC 成员实验室，我们广泛合作，以辨别动态平衡和损伤/储备的生物学 ISCS相对于它们的利基。在这里，我们继续这些研究的三个目标。 在目标1中，我们继续我们先前对Lgr5+ISC生态位的研究，以探索Lgr5+ISC之间的串扰 和其密切相关的PDGFRA+间充质细胞通过表达PDGFRA的细胞感知和 对Lgr5+ISC损伤作出反应，而Lgr5+ISC反之对PDGFRA+细胞产生刺激因子。这 扩展了初步数据，其中通过辐射或R-响应素对Lgr5+ISC进行一次操作会导致二次 对PDGFRA+细胞数量、增殖和包括R-Spinins在内的利基因子表达的影响。此Lgr5+ ISC/PDGFRA+串扰将通过删除PDGFRA+人群中的R-Spinins以及通过遗传和 血小板衍生生长因子信号的药理学操作。补充这一点的是肠样重建研究和 PDGFRA+细胞的无偏串联单细胞RNA-SEQ和ATAC-SEQ。 目的2进一步研究气液界面(ALI)肠组织中含有上皮细胞和 迈向临床前翻译的间质。首先，我们将通过IF来广泛地表征基质成分 并进行无偏单细胞rna-seq，以告知和优化ALI培养方法和基质。 保存在其中。其次，我们开展了人和小鼠ALI肠道的活体原位移植 有机化合物，利用生物兼容的ECM和第二代生物工程Wnt激动剂 具有FZD亚型特异性。第三，我们应用小鼠ALI有机化合物来实现第一个有机化合物 移植矫正肠道铁吸收铁蛋白(FPN)-KO模型中的疾病表型 缺乏性贫血。 最后，Aim 3利用我们的能力将生物工程Wnt激动剂转化为FZD亚型特异性 生物探针和治疗药物的拮抗剂。FZD亚型特异性拮抗剂将用于探测 体内肠道动态平衡过程中对特定FZD的需求，肠样培养平行建模。 总体而言，这些目标中的每一个都代表着高度协作的研究，如果没有完整的 ISCC实验室同事和ISCC协调中心的参与，以推进ISCC的双重目标 定义利基生物学和治疗性翻译。

Project Summary/Abstract The intestine is endowed with a remarkable ability to continually regenerate its epithelial layer, both to maintain tissue homeostasis and to heal injury, mediated by Lgr5-expressing active and injury/reserve intestinal stem cells (ISCs) and complex cellular and humoral niche. Disruption of these regenerative properties results in absorptive or barrier dysfunction or frank overgrowth in malabsorptive, infectious, inflammatory and neoplastic intestinal disorders. An improved understanding of ISCs and their niche is therefore crucial to the clinical translation of this biology as is the mission of the NIH Intestinal Stem Cell Consortium (ISCC). As a current ISCC member laboratory, we have extensively collaborated to discern the biology of homeostatic and injury/reserve ISCs relative to their niche. Here, we continue these studies in three aims. In Aim 1, we continue our prior studies on the Lgr5+ ISC niche to explore crosstalk between Lgr5+ ISC and its closely associated PDGFRA+ mesenchyme via the concept that PDGFRA-expressing cells sense and respond to Lgr5+ ISC damage, while Lgr5+ ISC conversely elaborate stimulatory factors for PDGFRA+ cells. This extends preliminary data where primary manipulation of Lgr5+ ISC by radiation or R-spondin results in secondary effects on PDGFRA+ cell number, proliferation and expression of niche factors including R-spondins. This Lgr5+ ISC/PDGFRA+ crosstalk will be studied by deletion of R-spondins in PDGFRA+ populations and by genetic and pharmacologic manipulation of PDGF signaling. This is complemented by enteroid reconstitution studies and unbiased tandem single cell RNA-seq and ATAC-seq of PDGFRA+ cells. Aim 2 furthers our studies of air-liquid interface (ALI) intestinal organoids containing epithelium and mesenchyme towards pre-clinical translation. First, we will extensively characterize stromal components by IF and perform unbiased single cell RNA-seq, to inform and optimize the ALI culture method and stromal preservation therein. Secondly, we develop in vivo orthotopic transplantation of human and mouse ALI intestinal organoids, leveraging biocompatible ECMs and leveraging second-generation bioengineered Wnt agonists having Fzd-subtype specificity. Thirdly, we apply mouse ALI organoids to achieve the first organoid transplantation correction of a disease phenotype in a Ferroportin (Fpn)-KO model of intestinal iron absorption deficiency anemia. Lastly, Aim 3 exploits our ability to convert bioengineered Wnt agonists into Fzd-subtype specific antagonists for biological probes and therapeutics. Fzd-subtype specific antagonists will be used to probe the requirement of specific Fzds during intestinal homeostasis in vivo, with parallel modeling in enteroid culture. Overall, each of these aims represent highly collaborative studies that would not be possible without the integral participation of fellow ISCC labs and the ISCC Coordinating Center, towards advancing the dual ISCC goals of defining niche biology and therapeutic translation.