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

项目摘要 世界上大约10%的成年人口患有慢性肾脏病(CKD)，这种疾病有非常严重的 很少有有效的预防或稳定治疗选择。此外，30%的新药没有 由于肾毒性而进展。我们已经开发了高效的定向微分协议来生成 来自人类多能干细胞(HPSCs)的肾单位祖细胞(NPC)和3D肾器官。在… 然而，目前还没有有效的平台来整合这些肾脏细胞和血管化的有机物质。 在体外微生理系统内建立有效的肾脏模型以研究肾毒性 和药效。我们的提案将肾脏器官和疾病、微生理系统、 以及由三名经验丰富的调查人员(Bonventre，Lee和Lewis)领导的生物打印，以独特的努力创建 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来指导差异化 HPSCs分化为肾小管足细胞、肾小管上皮细胞和血管内皮细胞 集成到微生理分析平台(MAP)和生物打印结构中的功能。我们创造了 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、建造和 描述完整的肾脏图谱以评估hPSC来源的肾脏足细胞、内皮细胞的功能 以及肾上皮细胞和肾脏器官。我们还将使用这个平台来创建一个模型 在细胞外基质上有分化足细胞的肾小球(模拟肾小球基底 膜)和hPSC来源的内皮细胞位于基底膜的另一侧。地图将会 优化询问基础肾脏生物学和非遗传性和遗传性疾病的病理生物学 涉及肾囊肿或足细胞损伤，以及对可能的治疗药物的测试反应。在特定目标中3 我们将制作一个包含曲折的近端小管、周细胞和内皮衬里的3D肾脏模型。 具有受控的、生理相关的系统的血管结构。模拟的小管和血管系统将是 通过开放的管腔灌流。ECM成分将被优化以支持融合上皮化 使用来自HiPSCs的近端和远端小管细胞、足细胞和内皮细胞。我们会 表征极化药物的摄取、毒性和通过间质(ECM)的载体运输以及 上皮细胞、间质和内皮细胞通道之间的细胞-细胞相互作用 验证由健康人和囊性疾病患者的细胞组成的带血管肾脏模型 这会影响影响足细胞的小管和肾小球疾病。我们的计划，有很好的历史 里程碑，将导致新的模型来测试肾脏毒性和药物疗效。

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.