肢芽前体细胞移植诱导小鼠近端断趾再生的方法及其机制研究

While mammals including humans are generally considered to have lost the capacity of regenerating an injured limb, the prospect of human limb regeneration is encouraged by the observation that human and mouse fingertips are able to regenerate. Interestingly this regeneration ability is dependent on the level of amputation. Only the extreme distal tip of human fingers can regenerate, while injuries in proximal digits all result in scar formation and no regeneration. This level dependence of digit regeneration provides an excellent model for exploring measures to stimulate regeneration. In our previous studies on Xenopus limb regeneration, we have for the first time succeeded in inducing frog limb regeneration by transplantation of limb progenitor cells to the limb stump. The success relies on the use of fibrin scaffold for cell delivery, which mimics the regeneration blastema in the limb stump; the activation of Wnt/beta-catenin signaling in the transplanted cells; and the provision of growth and patterning factors (Shh, Fgf10, thymosin b4), which promote cell survival and differentiation. To explore the possibility of using progenitor cell transplantation to stimulate mouse digit regeneration, we have modified the growth factors to be supplemented to the mouse limb progenitors to be used for transplantation, and established a transplantation approach that allows long-term survival of progenitors in the mouse digit stump. We have identified that Bmp2, Fgf8, Wnt3a and Thymosin b4 are good candidates for promoting proliferation and in vitro differentiation potential of isolated embryonic limb progenitor cells. In this proposal, we will systemically optimize the combinations of growth factors for mouse limb progenitor cell transplantation by analyzing digit regeneration in the adult mouse middle phalanges. We will also analyze the regeneration behaviors of the transplanted limb progenitor cells and the host limb tissues during stimulated regeneration, and investigate the molecular targets of the growth factors applied in the cell transplants. The findings from this study will provide critical insights on the development of new treatment for human limb and digit injuries in the future.

人类不能再生断肢，但能再生离断的末端指尖。这说明人类仍有再生断肢及断指的潜能。小鼠断趾再生与人类断指再生极其相似，是研究哺乳动物断指再生的理想模型。但目前尚无有效刺激小鼠断趾再生的手段。我们新近发现移植含有合适的生长因子和胚胎肢芽前体细胞的纤维蛋白骨架可以模拟再生芽基以有效诱导成体爪蟾断肢再生。为将该技术成功拓展到哺乳动物，本课题将对已筛到的Bmp2, Fgf8, Wnt3a及Thymosin b4四个因子进行优化组合，体外分析各组合对肢芽前体细胞增殖与分化的影响；以小鼠近端断趾为模型在体内用X线成像及免疫组化等手段分析断趾在不同因子组合加肢芽前体细胞移植的再生情况；分析前体细胞与断肢残端自身细胞相互作用；明确生长因子协助肢芽前体细胞促进断趾再生的机制。本课题将建立基于细胞移植诱导断指再生的技术平台，为探索利用多能细胞如iPS细胞来源的肢芽前体细胞诱导人类断肢再生提供重要的理论和实验依据。

尽管非常局限，人和小鼠的指/趾尖的确能够再生，这为诱导人类断指及断肢再生提供了理论上的可能性。但目前尚无有效刺激人断指或小鼠断趾再生的手段。在前期成功诱导成体爪蟾断肢再生的基础上，本项目研究了肢芽前体细胞移植诱导小鼠近端断趾的可能性及条件。我们确定了肢芽前体细胞体外扩增及移植所需的最近生长因子BFTW（Bmp2、Fgf8、Thymosin b4、Wnt3a）组合，以小鼠第三趾第二趾节（Digit 3 Phalange 2, D3P2）近端断趾损伤为模型证明了肢芽前体细胞+BFTW因子组合可以有效诱导成体小鼠近端断趾再生，分析了D3P2在肢芽前体细胞移植后的再生情况，证明了肢芽前体细胞不仅参与骨组织的再生，也参与了再生断趾中结缔组织的重建。我们也证明了生长因子对细胞在断趾末端移植后的存活及增殖是必须的。此外，本项目将肢芽前体细胞移植的实验在原计划基础上扩展到从iPSCs获得肢芽前体细胞并证明了iPSCs来源的类肢芽前体细胞可以诱导小鼠D3P2近端断趾再生。我们建立了从多能干细胞诱导分化肢芽类器官及类肢芽前体细胞的实验标准流程。这将为进一步探索多能干细胞来源的前体细胞移植在人类断肢损伤及器官再生中的转化应用提供了重要理论基础及实验依据。