Engineering induction and assembly of human kidney tissue

项目摘要 这项计划的目的是研究和控制肾单位的诱导和组装， 替代肾组织肾脏类器官重新创造了惊人的细胞多样性，可与 早期胎儿肾然而，产尿肾单位及其排水网络的结构连接性 由输尿管上皮（UE）形成的输尿管上皮细胞（UEs）需要避免快速病变，但尚未实现。因此，委员会认为， 因此迫切需要在肾移植之前实现肾单位和输尿管上皮之间的连接， 类器官可以在再生医学中发挥其潜力。我们的长远目标是构建'更高层次' 使用人类自体干细胞谱系和组装技术的合成肾组织， 形态发生的结果。我们在这个阶段的总体目标是首先获得对肾单位的空间控制 通过确定机械微环境如何影响其诱导部位来形成， 成熟它的第二个目标是通过一个受控的免疫反应，在许多空间位点指导肾单位与UE融合。 侵入性过程实现这些目标将标志着在创造替代品方面取得变革性进展。 肾脏组织我们的中心假设是，在肾脏发育过程中，间充质细胞的机械压实 形态发生允许肾单位诱导，随后是对WNT严格时空控制 信令事件对于它们与UE的有效融合是必要的。我们计划透过以下两方面达致目标： 具体目标。首先，我们将确定肾发生的力学基础，并用它来说明 肾单位位置。我们将研究肾源性间充质的机械压实，评估生物物理 早期肾单位细胞的特性，并使用微图案在特定位置优化肾发生 技术.其次，我们将设计WNT诱导的肾单位与输尿管上皮的融合。我们将 使用对WNT信号传导的光遗传学控制优化肾单位-输尿管上皮共培养物中的融合，然后 在将UE球状体从微图案化表面转移后，触发肾单位与UE球状体的组装。拟议 研究是创新的，因为我们在创造生物材料组织的同时创造了基础知识， 免费，人源性（与患者源性自体细胞策略兼容），因此对未来开放 发展移植。该研究具有重要意义，因为人类的高阶组装 肾脏组织将使肾脏替代技术发生飞跃性变化，超越透析、移植和“非生物” 过滤我们希望这些努力在基础生物学领域产生重大的积极影响， 发现、药物靶点筛选和再生医学。

PROJECT SUMMARY The goal of this proposal is to study and control nephron induction and assembly towards the formation of replacement renal tissue. Kidney organoids re-create an astonishing cellular diversity comparable to the early fetal kidney. However, structural connectivity of urine-producing nephrons and their drainage network formed by ureteric epithelium (UE) is required to avoid rapid pathology, yet has not been achieved. Accordingly, there is an urgent need to achieve connectivity between nephrons and ureteric epithelium before kidney organoids can achieve their potential in regenerative medicine. Our long-term goal is to construct ‘higher-order’ synthetic kidney tissues using human autologous stem cell lineages and assembly technologies that mimic the outcomes of morphogenesis. Our overall objectives at this stage are firstly to gain spatial control over nephron formation by determining how the mechanical microenvironment contributes to their induction sites and maturation. Its second objective is to direct nephron fusion with UE at many spatial sites through a controlled invasive process. Achieving these objectives will mark a transformative advance towards creating replacement kidney tissue. Our central hypothesis is that mechanical compaction of mesenchymal cells during kidney morphogenesis permits nephron induction, and subsequently that tight spatiotemporal control over WNT signaling events is necessary for their efficient fusion with UE. We plan to achieve the objectives through two specific aims. Firstly, we will determine the mechanical basis of nephrogenesis and use it to specify nephron positions. We will study mechanical compaction of the nephrogenic mesenchyme, assess biophysical properties of early nephron cells, and optimize nephrogenesis at specific locations using micropatterning technology. Secondly, we will program WNT-induced fusion of nephrons with ureteric epithelium. We will optimize fusion in nephron-ureteric epithelial co-cultures using optogenetic control over WNT signaling, and then trigger nephron assembly with UE spheroids after transferring them from micropatterned surfaces. The proposed research is innovative because we create fundamental knowledge while creating tissues that are biomaterial- free, human-derived (compatible with patient-derived autologous cell strategies), and therefore open to future development for transplantation. The proposed research is significant because higher-order assembly of human kidney tissue will create a step-change in renal replacement technology beyond dialysis, transplant, and “abiotic” filtration. We expect these efforts to have significant positive impact in the areas of fundamental biological discovery, drug target screening, and regenerative medicine.