Generation of human glomerular spheroids and filtration barrier in a vascularized milieu as a personolized model for glomerular diseases

在肾脏内，肾小球是尿液通过肾小球滤过屏障从血液中过滤的功能单位。这种屏障由有窗口的肾小球内皮细胞、肾小球基底膜和足细胞组成。足细胞受损可导致肾病综合征。足细胞基因突变是激素抵抗型肾病综合征(SRNS)的原因之一。在常染色体显性突变中，这种疾病通常出现在年轻的成年期，这意味着第二次发作。SRNS被认为对免疫抑制治疗无反应，并进展为终末期肾脏疾病。然而，最近的数据表明，在某些情况下，环孢素A和类固醇的组合可以导致缓解。这种治疗概念的机制在很大程度上是未知的。多年来，细胞培养模型一直被用于体外研究肾小球疾病。然而，由于缺乏3D背景、不同细胞类型之间的肾小球干扰、缺乏患者来源的细胞以及血管化的环境，这些模型与自然肾小球的相似性有限。本项目旨在建立肾小球结构，以更好地了解肾小球在生理和病理生理条件下的功能和相互作用。因此，我们将在体外将不同个性化的IPSC来源的人足细胞、人肾小球内皮细胞和系膜细胞共同培养成肾小球球体，然后在我们建立的房室环大鼠模型中将这些结构血管化。此外，我们还将患者来源的足细胞与人肾小球内皮细胞一起培养在人工基底膜的两个位置上，并在我们的房室环大鼠模型中将该人工基底膜血管化。为了产生患者的IPSC足细胞，我们将使用WT1和INF2突变患者以及健康志愿者的皮肤成纤维细胞，将其重新编程为IPSC，并将其分化为保持患者突变的足细胞。最后，我们将移植血管模型，并将它们用于不同的治疗和毒性筛选。血管共培养模型的单细胞序列分析和形态特征将允许在治疗和不治疗的其他肾小球细胞的背景下分析患者的足细胞。因此，我们希望以患者个性化的方式测试SRNS的治疗选择以及对有毒物质的敏感性。

Within the kidney, the glomerulus is the functional unit where the urine is filtered from the blood through the glomerular filtration barrier. This barrier consists of fenestrated glomerular endothelial cells, the glomerular basement membrane and podocytes. Impairments in podocytes can lead to nephrotic syndrome. Mutations in podocyte gens is a cause for steroid resistant nephrotic syndrome (SRNS). In autosomal dominant mutations, the disease usually manifest in young adulthood suggesting a second hit. SRNS is believed to not response to immunosuppressive therapy and to progress to end stage renal disease. However, recent data suggest that a combination of cyclosporine A and steroids can lead to remission in some cases. Mechanisms of this therapeutic concept are largely unknown. For years, cell culture models have been used to study glomerular diseases ex vivo. However, these models are limited in their resemblance to nature glomeruli due to lack of 3-D context, glomerular crosstalk between different cell types, absence of patient derived cells, and a vascularized milieu. This project aims to establish glomerular structures to better understand glomerular functions and interaction under physiological and pathophysiological conditions. Therefore, we will generate glomerular spheroids with different personalized iPSC derived human podocytes, human glomerular endothelial cells and mesangial cells in co-culture ex vivo and later vascularize these structures in our established AV loop rat model.Furthermore, we will culture patient-derived podocytes together with human glomerular endothelial cells on two sites of an artificial basement membrane and vascularize this artificial basement membrane in our AV loop rat model. To generate iPSC podocytes of patients we will use skin fibroblast of patients with mutations in WT1 and INF2 as well as healthy volunteers, reprogram them into iPSCs and differentiate them into podocytes keeping the patients mutation. Finally, we will explant the vascularized models and use them for different therapeutic and toxic screenings. Single cell seq analysis and morphological characterization of the vascularized co-culture models will allow analyzing the patient derived podocytes in the context of other glomerular cells with and without treatment. Herewith, we want to test therapeutic options in SRNS as well as susceptibility towards toxic substances in a patient-individualized manner.