Kidney Microphysiological Analysis Platforms (MAP) to Optimize Function and Model Disease

项目摘要 世界上大约10%的成年人患有慢性肾病（CKD）， 很少有有效的预防或稳定治疗选择。此外，30%的新开发药物 因为肾毒性而恶化我们已经开发了有效的定向分化方案， 肾单位祖细胞（NPC）和来自人多能干细胞（hPSC）的3D肾类器官。在 然而，目前还没有整合这些肾细胞和血管化类器官的有效平台， 在体外微生理系统中开发有效的肾脏模型，用于询问肾毒性 和药物功效。我们的提案结合了肾脏类器官和疾病，微生理系统， 和生物打印由三位经验丰富的研究人员（Bonventre，Lee和刘易斯）领导， 这些需要的模型平台。在具体目标1中，我们将开发有效的流程， hiPSC转化为肾足细胞、肾小管上皮细胞和内皮细胞，具有分化的 集成到微生理分析平台（MAP）和生物打印结构中的功能。我们创造 疾病的遗传模型和指示分化特征以优化分化的报告系 协议和监测生理参数。在具体目标2中，我们将设计，建造， 表征整合的肾脏MAP以评价hPSC衍生的肾脏足细胞、内皮细胞和内皮细胞的功能。 和上皮细胞以及肾脏类器官。我们还将利用这个平台创建一个模型， 在细胞外基质上具有分化的足细胞的肾小球（以模拟肾小球基底 在基底膜的另一侧是hiPSC衍生的内皮细胞。地图会 优化以询问非遗传和遗传疾病的基本肾脏生物学和病理生物学 涉及肾囊肿或足细胞损伤和测试对推定治疗剂的反应。具体目标3 我们将生物打印一个3D肾脏模型，该模型包含近端曲小管、周细胞和内皮细胞。 具有受控的生理相关系统的血管结构。模拟的小管和脉管系统将 通过开放的管腔灌注。将优化ECM组合物以支持融合上皮形成 使用来源于hiPSC的近端和远端小管细胞、足细胞和内皮细胞。我们将 表征极化药物摄取、毒性和通过细胞外基质（ECM）的载体转运，以及 上皮细胞、上皮细胞和内皮细胞衬里的通道之间的细胞-细胞相互作用， 验证由来自健康和囊性疾病患者的细胞组成的血管化肾脏模型 影响肾小管和肾小球的疾病影响足细胞。我们的计划，拥有完善的 里程碑，将导致新的模型来测试肾毒性和药物疗效。

Project Summary Approximately 10% of the world's adult population has chronic kidney disease (CKD) for which there are very few effective preventive or stabilizing therapeutic options. In addition, 30% of newly developed drugs are not advanced because of nephrotoxicity. We have developed efficient directed differentiation protocols to generate nephron progenitor cells (NPCs) and 3D kidney organoids from human pluripotent stem cells (hPSCs). At present, however, there are no effective platforms that integrate these kidney cells and vascularized organoids within microphysiological systems in vitro to develop effective kidney models for interrogation of nephrotoxicity and drug efficacy. Our proposal unites expertise in kidney organoids and disease, microphysiological systems, and bioprinting led by three experienced investigators (Bonventre, Lee and Lewis) in a unique effort to create these needed model platforms. In Specific Aim 1 we will develop efficient processes to direct differentiation of hiPSCs into kidney podocytes, tubular epithelial cells, and endothelial cells endowed with differentiated features for integration into microphysiological analysis platforms (MAP) and bioprinted structures. We create genetic models of disease and reporter lines that signal differentiation characteristics to optimize differentiation protocols and to monitor physiological parameters. In Specific Aim 2 we will design, construct, and characterize an integrated kidney MAP to evaluate the function of hPSC-derived kidney podocytes, endothelial and epithelial cells as well as kidney organoids. We will also use this platform to create a model of a glomerulus that will have differentiated podocytes on an extracellular matrix (to mimic the glomerular basement membrane) and hiPSC-derived endothelial cells on the other side of the basement membrane. The MAP will be optimized to interrogate basic kidney biology and pathobiology of both non-genetic and genetic disease involving kidney cysts or podocyte injury and test responses to putative therapeutic agents. In Specific Aim 3 we will bioprint a 3D kidney model that contains convoluted proximal tubules, pericytes and endothelial-lined vascular structures with controlled, physiologically relevant system. Modeled tubules and vasculature will be perfused through a open lumens. The ECM composition will be optimized to support confluent epithelialization using proximal and distal tubule cells, podocytes, and endothelial cells derived from hiPSCs. We will characterize polarized drug uptake, toxicity, and vectorial transport through the interstitium (ECM) as well as cell-cell interactions among the epithelial cells, interstitium and endothelium-lined channels to create and validate vascularized kidney models composed of cells derived from healthy and patients with cystic disease that affects the tubule and glomerular disease that affects the podocyte. Our program, with well-established milestones, will result in novel models to test kidney toxicity and drug efficacy.