如何使受损和衰老组织再生出有功能的新组织，一直是生物学和临床医学面临的重大难题之一。涡虫已成为研究再生生物学的重要模式动物之一，涡虫体内的成体多功能干细胞是涡虫强大再生能力的细胞学基础，但这些多功能干细胞的分子标签还有待进一步鉴定，也没有成功的体外培养方法。申请人近期的研究发现，一种新的DNA染料SiR-DNA可以富集涡虫体内可增殖分化的细胞，命名为SirNeoblasts，同时，申请人也建立了维持涡虫干细胞多能性的体外培养方法，因此，申请人将通过本研究鉴定和纯化SirNeoblasts中的多功能干细胞，并且优化涡虫干细胞体外增殖的方法。申请人将首先定量分析SirNeoblasts的多能性和异质性，然后通过单细胞转录组分析和细胞移植实验，鉴定SirNeoblasts中多功能干细胞，最后确定可促进涡虫成体干细胞体外增殖的细胞因子。本研究对理解涡虫再生的细胞学基础有重要意义。

Planarians are widely used research organisms in regenerative biological studies. They possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole–body regeneration. However, the identity and culture methodology of pluripotent neoblasts are currently unavailable. Our recent efforts have identified a new DNA-staining dye SiR-DNA, which can enrich the proliferative cells named SirNeoblasts from planarians. Meanwhile, we have also developed a cell culture method to maintain the pluripotent stem cells in vitro. We therefore apply for identifying and purifying the pluripotent stem cells from SirNeoblasts, as well as optimizing our cell culture method for cell proliferation in vitro. First, we will quantitatively analyze the cellular pluripotency and heterogeneity in SirNeoblasts. Second, we will perform the single-cell RNA sequencing and single cell transplantation to identify the pluripotent stem cells in planarians. Third, we will utilize SirNeoblasts and/or purified planarian pluripotent stem cells to examine the function of different growth factors in promoting cell proliferation in culture. This research will significantly advance our understanding on the cellular basis of planarian regeneration and regenerative biology.