Exploiting skin fibroblasts to promote digit tip regeneration upon amputation

总结 仅在美国就有近200万人失去了肢体。迄今为止， 人类肢体再生的能力然而，远端肢体区域，手指尖，可以经历完美的 再生哺乳动物指端再生是表型再生的一个显著例子。后 在小鼠和人类的趾尖截肢后，未分化的间充质，称为芽基， 在伤口表皮下，通过一个组织学上反映两栖动物肢体再生的过程。这 芽基最终分解成完全再生的尖端，包括指甲和骨头。虽然这种再生 能力仅限于手指的远端，理解这一现象可以提供关于 近端截肢的再生。 目前，对哺乳动物的关键结构芽基的起源和特征知之甚少 成功的指尖再生。截肢残端的细胞在多大程度上经历了脱- 在芽基形成和随后的再生过程中的分化/转分化仍然是难以捉摸的 在低等脊椎动物中，甚至在哺乳动物中也是未知的。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芽基，然后改变它们的命运进入骨谱系， 茎尖再生这提出了一个令人兴奋的可能性，即成纤维细胞，人体内丰富的细胞，可能具有 创造再生能力芽基的潜力。 我们假设外源性成纤维细胞有能力产生芽基， 分化成新的趾骨和周围的间充质。解决这一假设的一个主要挑战是 以及其他关于尖端再生的基本问题，是该领域众所周知的警告， 小费是非常小的。我们提出了一个逐步的项目：1）探索使用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 大于鼠标尖端）。在R61阶段，我们将全面表征大鼠指尖再生 使用组织化学和scRNA测序，并开始测试外源性成纤维细胞经历 移植到足趾尖时的骨线转化。我们将追踪成纤维细胞的行为， 确定它们对大鼠远端和近端截肢后指尖再生的贡献。在R33中 阶段，我们将从大鼠手指分离胚基细胞，并检查胚基细胞发育过程中观察到的表观遗传变化。 在大鼠中使用ATAC seq.同时，我们将测试外源性人成纤维细胞如何促进指尖 使用免疫缺陷大鼠作为受体进行再生。这些实验将有助于建立有效的细胞 创造或至少促进哺乳动物胚基形成的治疗方法，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.