基于单细胞高通量解析和类器官精确构建的子宫发育与再生研究

The serious injury of endometrium and smooth muscle caused by clinical common diseases and operations would be too difficult to repair and regenerate, even lead to infertility but have rare effective treatment. At present, there is a lack of knowledge about cell composition and the regulation of differentiation in the development of embryonic uterus，this study aims to analyze cell subsets and the regulatory mechanism of differentiation, guiding the precise construction of uterine organoid and uterine regeneration. We would use high-throughput single cell analysis to detect the cell atlas and signal pathway in the development process of mouse uterus, establish induced differentiation culture system of human induced pluripotent stem cells (hiPSC)-derived endometrial epithelial progenitor cells and stromal progenitor cells in vitro, combined with hiPSC-derived smooth muscle progenitor cells, take gelatin methacrylate (GelMA) as the print "ink" to precisely construct the whole layer uterine organoid by sterolithography apparatus（SLA）technology, for repairing and regenerating the whole layer injury of the uterus. This study would reveal the cell atlas and signal regulatory pathway of uterine development, obtain new knowledge of inducing hiPSC to differentiate into endometrial epithelial/ stromal progenitor cells in vitro, and establish a new tissue engineering strategy of uterine repair and regeneration.

临床上常见疾病和手术导致子宫内膜和肌壁严重损伤，修复与再生困难，引起不孕且治疗困难。目前子宫的细胞构成和分化调控知识缺乏，本研究拟高清解析子宫发育过程的细胞亚群构成和分化调控机制，指导子宫类器官的精准构建和子宫再生。本研究将通过单细胞高通量测序解析小鼠子宫发育过程的细胞图谱和信号调控通路，探索建立人诱导性多能干细胞（hiPSC）定向诱导分化为子宫内膜上皮前体细胞和基质前体细胞的体外培养条件，联合hiPSC来源的平滑肌祖细胞，利用甲基丙烯酸酯明胶（GelMA）为打印“墨水”，借助光固化生物打印技术进行全层子宫类器官精准构建，开展子宫全层修复与再生的效应研究。本研究将揭示子宫发育过程的细胞图谱和信号调控通路；获得体外诱导hiPS向子宫内膜上皮前体细胞/基质前体细胞稳定分化新知识；建立子宫修复再生的组织工程新手段。

子宫内膜损伤是女性生殖系统常见疾病之一，严重的内膜损伤后修复与再生困难，导致不孕，临床上仍然没有有效的预防或治疗方法。人多能干细胞（hPSCs，human pluripotent stem cells）的诱导分化以及类器官的构建，在再生医学中展现广阔的应用前景，但目前尚缺乏精准、高效诱导hPSCs向子宫内膜分化的方法。分化研究最好的借鉴模板就是组织发育过程，然而我们对子宫发育的认识还很有限。因此，本研究针对以上问题，1）首先运用单细胞转录组测序（scRNA-seq）技术解析了雌性小鼠的子宫前体—苗勒管（MD，Müllerian Duct）的发育，绘制发育过程细胞图谱和信号通路；2）通过调控信号通路，模拟该发育过程中分化的路径，体外培养条件下定向诱导hPSCs向苗勒管样细胞分化，构建功能性人子宫内膜类器官；3）探索hPSCs来源的苗勒管样细胞用于功能性修复大鼠子宫内膜全层损伤；4）同时开展3D生物打印技术构建双层子宫内膜类器官，评估其修复全层子宫损伤的能力。本研究成果通过scRNA-seq对苗勒管发育过程中的细胞、分子等有更清晰的了解，开拓了研究胚胎期子宫发育的新局面；建立了简单、明确、有效的诱导方案，获得hPSCs分化来源的苗勒管样细胞，证实了其双向分化潜能（内膜上皮、内膜基质）和修复大鼠子宫内膜损伤的能力；结合3D生物打印精准构建出仿生双层的子宫内膜类器官展现出更好的功能性修复效果，为将来治疗子宫内膜损伤疾病提供一种有潜力的种子细胞和组织工程策略。本项目成果达到结题预期，发表SCI论文11篇（其中有6篇论文影响因子大于10分），申请国家发明专利1项，培养博士后1名、博士2名、硕士1名。