Exploiting skin fibroblasts to promote digit tip regeneration upon amputation

摘要 仅在美国就有近200万人生活在失去肢体的情况下。到目前为止，还没有办法推广 人类四肢再生的能力。然而，远端肢体区域，即指尖，可以经历完美的 再生。哺乳动物的指尖再生是表型再生的一个显著例子。vt.在.的基础上 在小鼠和人的指尖截断，未分化的间质，称为胚基，形成 在伤口表皮下，通过组织学上反映两栖动物肢体再生的过程。这 胚泡最终会分解成一个完美再生的尖端，包括指甲和骨头。尽管这种再生 能力仅限于指尖的远端，理解这种现象可以提供关于 更多近端截肢的再生。 目前，人们对哺乳动物胚泡的起源和特征知之甚少。 对于成功的指尖再生。截肢残端的细胞在多大程度上经历了降解- 胚泡形成和随后的再生过程中的分化/转分化仍然难以捉摸 在低等脊椎动物中，事实上，在哺乳动物中是未知的。我们在小鼠身上的初步结果表明，成纤维细胞可以 迁移到截肢部位形成胚泡，然后改变他们的命运进入骨谱系以协助 尖端再生。这提出了一种令人兴奋的可能性，即人体内丰富的成纤维细胞可能具有 创造出具有再生能力的胚泡的潜力。 我们假设外源性成纤维细胞有能力产生胚泡，最终 分化为新的指骨和周围的间质。解决这一假说的一个主要挑战 和其他关于尖端再生的基本问题，是该领域众所周知的告诫，老鼠指的是 小费非常小。我们提出了一个循序渐进的计划：1)探索将大鼠作为临床前研究的可行性 指尖再生模型；2)将大鼠(可能还有人)成纤维细胞移植到大鼠指尖(30次) 比鼠标尖大)。在R61阶段，我们将全面描述大鼠指尖再生的特征 使用组织化学和scRNA序列，并开始检测外源成纤维细胞经历 移植到指尖时的骨线转换。我们将追踪成纤维细胞的行为 确定它们在大鼠远端和近端截肢后指尖再生中的作用。在R33中 阶段，我们将从大鼠的指头分离胚泡细胞，并检查胚泡期间所见的表观遗传学变化 在使用ATAC序列的大鼠中形成。同时，我们将测试外源人类成纤维细胞如何促进指尖 以免疫缺陷大鼠为受体进行再生。这些实验将有助于建立一个有效的细胞 创造或至少促进哺乳动物胚芽形成的疗法，最终用于肢体再生。

Summary Nearly 2 million people live with a lost limb in the United States alone. To date, there is no way to promote the ability to regenerate limbs in humans. Yet the distal limb region, the digit tip, can undergo perfect regeneration. Mammalian digit tip regeneration is a remarkable example of epimorphic regeneration. Upon amputation of the digit tip in mice and humans, undifferentiated mesenchyme, called the blastema, forms underneath the wound epidermis, through a process that histologically mirrors amphibian limb regeneration. This blastema ultimately resolves into a perfectly regenerated tip, including nail and bone. Although this regeneration ability is limited to the distal tip of the digits, understanding this phenomenon can provide unique insights about regeneration of more proximal amputations. Currently, little is known about the origin and characteristics of the mammalian blastema, the key structure for the successful digit tip regeneration. To what extent the cells at the amputation stump undergo de- differentiation/trans-differentiation during the blastema formation and subsequent regeneration remains elusive in lower vertebrates and indeed, unknown in mammals. Our preliminary results in mice show that fibroblasts can migrate into the amputation site to form the blastema, and then alter their fate into the bone lineage to assist in tip regeneration. This raises an exciting possibility that fibroblasts, abundant cells within human body, may have the potential to create a regeneration-competent blastema. We hypothesize that exogenous fibroblasts are competent to give rise to blastema that ultimately differentiates into new digit bone and surrounding mesenchyme. A major challenge in addressing this hypothesis and other essential questions about tip regeneration, is the well-known caveat in the field that the mouse digit tip is extremely small. We propose a step-wise project: 1) Explore the feasibility of using rats as a preclinical model for digit tip regeneration; 2) transplant rat (and possibly human) fibroblasts into the rat digit tip (30 times larger than mouse tip). During the R61 phase, we will comprehensively characterize the rat digit tip regeneration using histochemistry and scRNA seq, and begin testing the ability of exogenous fibroblasts to undergo osteolineage conversion upon transplantation into the digit tip. We will trace the behavior of fibroblasts to determine their contribution to the digit tip regeneration upon distal and proximal amputations in rats. In R33 phase, we will isolate blastemal cells from rat digits and examine epigenetic changes seen during the blastemal formation in rats using ATAC seq. In parallel, we will test how exogenous human fibroblasts promotes digit tip regeneration using immunodeficient rats as recipients. These experiments will help establish an effective cellular therapy to create, or at the least, promote blastema formation in mammals ultimately for limb regeneration.