Investigation and Translation of the Intestinal Stem Cell Niche

项目总结/摘要 肠被赋予了一种非凡的能力，可以不断地再生其上皮层， 组织稳态和愈合损伤，由表达Lgr 5的活性和损伤/储备肠干介导 细胞（ISCs）和复杂的细胞和体液生态位。这些再生特性的破坏导致 在吸收不良、感染性、炎症性和肿瘤性中的吸收或屏障功能障碍或明显过度生长 肠道疾病因此，对ISC及其利基的更好理解对于临床治疗至关重要。 这是NIH肠道干细胞联盟（ISCC）的使命。作为现任ISCC 成员实验室，我们已经广泛合作，以辨别生物稳态和损伤/储备 ISCs相对于其利基。在这里，我们继续这些研究在三个目标。 在目标1中，我们继续之前对Lgr 5 + ISC利基的研究，以探索Lgr 5 + ISC之间的串扰 及其紧密相关的PDGFRA+间充质，通过PDGFRA表达细胞感知和 响应Lgr 5 + ISC损伤，而Lgr 5 + ISC相反地阐述PDGFRA+细胞的刺激因子。这 扩展了初步数据，其中通过辐射或R-spondin对Lgr 5 + ISC的主要操纵导致继发性 对PDGFRA+细胞数量、增殖和包括R-spondins在内的生态位因子表达的影响。LGR 5 + ISC/PDGFRA+串扰将通过PDGFRA+群体中R-脊椎蛋白的缺失和通过遗传和 PDGF信号传导的药理学操作。这是补充肠重建研究， 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）肠类器官， 间充质向临床前转化。首先，我们将通过IF广泛表征基质成分， 并进行无偏的单细胞RNA-seq，以告知和优化ALI培养方法和基质 其中，第二，我们建立了人和小鼠ALI小肠原位移植模型， 类器官，利用生物相容性ECM和利用第二代生物工程Wnt激动剂 具有FZD亚型特异性。第三，我们应用小鼠ALI类器官来获得第一类器官 在肠铁吸收的膜铁转运蛋白（Fpn）-KO模型中移植校正疾病表型 贫血症。 最后，Aim 3利用我们的能力将生物工程化的Wnt激动剂转化为Fzd亚型特异性受体。 用于生物探针和治疗剂的拮抗剂。FZD亚型特异性拮抗剂将用于探测 在体内肠内稳态过程中特异性Fzd的需求，在肠样培养中进行平行建模。 总的来说，这些目标中的每一个都代表了高度合作的研究，如果没有整合， ISCC实验室和ISCC协调中心的参与，以推进ISCC的双重目标， 定义生态位生物学和治疗翻译。

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.