Extracellular Matrix Induction of Renal Stem and Progenitor Cell Development

 描述(由申请人提供)： 急性肾脏损伤(AKI)和慢性肾脏疾病(CKD)是退伍军人社区的严重健康问题。在损伤后缺乏足够的肾小管修复的情况下，尽管有最好的药物治疗，肾功能下降通常会导致透析和显著的发病率和死亡率。日益增长的可移植器官需求和有限的供应之间日益扩大的差距，现在是阻碍肾移植扩大到所有终末期肾病患者和退伍军人的主要限制障碍。干细胞生物学，特别是诱导多能干细胞(Ipsc)技术，通过基因组和个体化医学的创新进展提供了新的策略，以破译肾脏修复的机制和影响组织再生的途径，为未来的临床干预修复提供机会。 损伤肾小管或替换受损的肾组织。我们解决了干细胞技术目前的局限性--描述和靶向三维(3D)组织中干细胞成熟的机制。为了确定这些基本参数，我们从去细胞的啮齿动物肾脏中开发并鉴定了无细胞3D支架，这些支架可以研究干细胞和前体细胞的生长和向后肾间充质分化，最终形成成熟的肾脏谱系。来自我们团队和其他人的证据表明，来自3D细胞外基质(ECM)支架的信号可以诱导干细胞成熟。我们的发现显示干细胞的分化具有地域特异性，E-钙粘蛋白的成熟和表达集中在基质衬里的小管上。我们的3D支架具有生物活性，由天然组织特异性成分组成 细胞外基质含有结构蛋白(如胶原蛋白、层粘连蛋白)和生长因子(如血管内皮生长因子、碱性成纤维细胞生长因子)，可介导肾脏器官发生。我们将检验这一假设，即来自3D肾脏ECM组织的信号是引导适当的干细胞和肾脏前体细胞分化形成肾单位节段的先决条件。肾脏的细胞复杂性促使我们特别关注无细胞基质支架内的小管生成，并确定体外再生和组织形成的要求。我们将使用两种肾脏干/祖细胞群：一种是早期肾来源的成年肾祖细胞，能够嵌入发育中的肾单位；另一种是多能的可重新编程的人类ipscs和胚胎干细胞群，它们分化形成早期的后肾组织。我们使用3D支架和干细胞的总体目标是阐明驱动分化的干细胞-基质相互作用的元素1将在使用小型ECM支架的高通量系统中研究干细胞/祖细胞对肾脏ECM修饰的反应，以快速筛选有利于分化的条件。在这个过程中，我们确定了必要的基质结合生长因子，并测试了 肾间质成纤维细胞对细胞外基质重塑的作用。目的2将通过分析较大器官大小的支架中对干细胞/祖细胞生长的要求来评估可伸缩性，并探索灌流生物反应器中的小管形成。该系统作为完整肾脏支架内肾单位发育的模型，并建立了干细胞分化和支架在正常和病变的ECM中再生的要求。这一研究策略使用创新的基质技术，用成纤维细胞和生物活性配体修饰组织支架，以描述3D分化的机制。我们的系统研究具有非常重要的意义，因为它将破译参与肾单位重建的关键因素，这是肾小管修复和肾组织再生的关键下一步。

 DESCRIPTION (provided by applicant): Acute kidney injury (AKI) and chronic kidney disease (CKD) are critical health problems in the Veteran community. In the absence of sufficient tubule repair after injury and despite the best medical therapy, decreasing kidney function often leads to dialysis and significant morbidity and mortality. The growing gap between the increasing demand and limited supply of transplantable organs is now the chief limiting obstacle preventing extension of kidney transplantation to all patients and veterans with end-stage renal disease. Stem cell biology, and in particular induced pluripotent stem cell (iPSC) technology, provides new strategies via innovative advances in genomic and personalized medicine to decode the mechanisms of renal repair and pathways influencing tissue regeneration, to offer an opportunity for future clinical intervention to repair injured renal tubules or replace failed renal tissue. We address the current limitation in stem cel technology-to delineate and target mechanisms of stem cell maturation in three-dimensional (3D) tissue. To define these fundamental parameters we developed and characterized acellular 3D scaffolds from decellularized rodent kidneys, which allow study of growth and differentiation of stem and progenitor cells toward metanephric mesenchyme, and ultimately a mature renal lineage. Evidence from our group, as well as others, indicate that signals from 3D extracellular matrix (ECM) scaffolds induce stem cell maturation. Our findings show stem cell differentiation in a geographically-specific manner with maturation and expression of E-cadherin centered around matrix-lined tubules. Our 3D scaffolds are bioactive, consisting of natural tissue- specific ECM with structural proteins (e.g. collagens, laminins) and growth factors (e.g. VEGF, bFGF) known to mediate renal organogenesis. We will test the hypothesis that signals from 3D renal ECM tissue are a prerequisite to direct proper stem and renal progenitor cell differentiation to form nephron segments. The cellular complexity of the kidney prompts us to specifically focus on tubulogenesis within acellular matrix scaffolds and determine the requirements for regeneration and tissue formation in vitro. We will use two renal stem/progenitor populations: an early kidney-derived, adult renal progenitor cell capable of intercalating into developing nephrons, and a population of pluripotent reprogrammed human iPSCs and embryonic stem cells that differentiate to form early metanephric tissue. Our overall goal using 3D scaffolds and stem cells is to elucidate the elements of stem cell-matrix interactions that drive differentiation Aim 1 will investigate stem/progenitor cell response to modification of the renal ECM in a high-throughput system using small ECM scaffolds to rapidly screen conditions favoring differentiation. In this process we identify the requisite matrix-bound growth factors and test the role of ECM remodeling by renal stroma fibroblasts. Aim 2 will assess scalability by analyzing the requirements for stem/progenitor growth in larger-scale organ-sized scaffolds and explore tubule formation within a perfusion bioreactor. This system serves as a model for nephron development within a full-scale kidney scaffold and establishes the requirements for stem cell differentiation and scaffold repopulation within normal and diseased ECM. This investigative strategy uses innovative matrix technology to modify tissue scaffolds with fibroblasts and bioactive ligands to delineate mechanisms of differentiation in 3D. Our systematic investigation is highly significant because it will decode the critical factors involved in nephron reconstitutio, which are critical next steps in tubule repair and renal tissue regeneration.