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

在肾脏中，肾小球是尿液通过肾小球滤过屏障从血液中过滤出来的功能单位。该屏障由开窗肾小球内皮细胞、肾小球基底膜和足细胞组成。足细胞损伤可导致肾病综合征。足细胞基因突变是类固醇抵抗性肾病综合征（SRNS）的一个原因。在常染色体显性突变中，这种疾病通常在年轻的成年期表现出来，这表明有第二次袭击。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和类固醇联合使用可导致缓解。这种治疗概念的机制在很大程度上是未知的。多年来，细胞培养模型已被用于体外研究肾小球疾病。然而，由于缺乏三维背景、不同细胞类型之间的肾小球串扰、缺乏患者来源的细胞和血管化的环境，这些模型在与天然肾小球的相似性方面受到限制。本项目旨在建立肾小球结构，以更好地了解生理和病理生理条件下肾小球的功能及其相互作用。因此，我们将用不同的个性化iPSC衍生的人足细胞、人肾小球内皮细胞和系膜细胞在体外共培养中产生肾小球球体，然后在我们建立的AV环大鼠模型中使这些结构血管化。此外，我们将在人造基底膜的两个部位培养患者衍生足细胞和人肾小球内皮细胞，并在我们的AV环大鼠模型中使人造基底膜血管化。为了生成患者的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，将其重编程为iPSC并将其分化为足细胞，保持患者的突变。最后，我们将移植血管化模型，并将其用于不同的治疗和毒性筛选。单细胞序列分析和血管化共培养模型的形态学特征将允许在其他肾小球细胞治疗和不治疗的背景下分析患者衍生的足细胞。因此，我们希望以患者个性化的方式测试SRNS的治疗方案以及对有毒物质的易感性。

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.