利用肾脏脱细胞支架诱导hiPSC来源的特定胚层细胞进行再生肾脏的构建及其机制研究

Kidney regeneration is the best way for end-stage renal disease. The technology based on the combination of renal scaffold and seeding cells provided new approach for this problem. However, some problems such as poor cell attachment, incomplete recellularization, cell polarity disorder and inflammatory reaction could be still found in the process of scaffold recellularization. Among them, difficult cell attachment and directional differentiation are the major problems. And there are also many problems in the study of kidney organoids with hiPSC, such as lack of blood supply and differential signals, low differential efficiency, etc. So we plan to construct tissue-engineered kidney by inducing two kinds of defined mesoderm cells which are co-cultured in decellularized kidney scaffold. Using the PCR, immunohistochemistry, WB, Micro-CT and some other methods to make a comprehensive evaluation of renal cells maturity, endothelial system tecontruction. And we could demonstrate that the co-culture system could promote the differentiation and formation of kidney, and unveil the mechanism of the specific core signaling pathways of kidney development and regeneration. The ultimate goal is to obtain a well-functional and transplantable tissue-engineered kidney. The implementation of this research will not only enrich the theory of renal regeneration and provide a theoretical basis for solving the problems of attachment and differentiation of stem cells in acellular scaffold, but also lay the technological foundation and provide the theoretical basis for the clinical transformation of renal regeneration.

肾脏再生是解决终末期肾病的最佳方法。基于脱细胞支架结合种子细胞技术为肾脏再生提供了新的研究方法。但在我们前期研究中证实，支架再细胞化过程存在原始干细胞分化效率低等问题。而应用hiPSC诱导至特定阶段，移植入体内，结合体内血管系统长入后，干细胞分化效率得到提高。因此我们提出假设，支架结合特定胚层细胞（肾系及血管内皮方向），通过支架的3D培养系统，不同胚层细胞相互作用能够提高干细胞在支架内的分化效率。因此本项目组拟采用支架与hiPSC来源的特定胚层细胞共培养构建再生肾脏，通过PCR、免疫荧光、WB、Micro-CT等方法研究肾系细胞分化成熟程度、内皮系统重建程度等，并从肾脏发育的特异性信号通路（Wnt）角度揭示支架对hiPSC来源的特定胚层细胞的诱导分化机制，获得功能良好的再生肾脏。本研究的实施将丰富肾脏再生理论，为解决干细胞在脱细胞支架的分化问题提供理论基础，并为肾脏再生的临床转化奠定基础。

肾脏再生是解决终末期肾病的最佳方法。基于脱细胞支架结合种子细胞技术为肾脏再生提供了新的研究方法。但在我们前期研究中证实，支架再细胞化过程存在原始干细胞分化效率低等问题。而应用hiPSC诱导至特定阶段，移植入体内，结合体内血管系统长入后，干细胞分化效率得到提高。本项目基于干细胞结合肾脏脱细胞支架构建再生肾脏，旨在研究肾系细胞分化成熟程度、内皮系统重建程度等，并从肾脏发育的特异性信号通 路(Wnt)角度揭示支架对hiPSC来源的特定胚层细胞的诱导分化机制，获得功能良好的再生肾脏。在项目实施过程中，我们采用诱导多能干细胞（IPSCs）诱导至间介中胚层（IM）及侧板中胚层阶段，结合肾脏脱细胞支架构建再生肾脏。我们成功应用IPSCs诱导至IM及侧板中胚层阶段，进行细胞种植，但肾脏再生过程中需要种子细胞数量较多，诱导的细胞数量难以维持供应，因此我们更改研究方案，将IPSCs分别诱导IM及类肾器官阶段，结合支架构建再生肾脏。通过研究，我们成功将IPSCs诱导至IM及类肾器官阶段，将其通过输尿管注入肾脏，血管区应用脐静脉内皮细胞进行种植，研究了IM及类肾器官在支架内的增殖、分化情况，同时研究了血管内皮与IM及类肾器官的相互作用。研究发现，类肾器官在支架内能够进一步分化成熟，提高了类肾器官诱导效率，并证实应用类肾器官能够构建再生肾脏。在此研究基础上，我们进一步将肾脏解剖至肾单位阶段，并构建了脱细胞支架，构建肾单位支架。