Tracing the origin of regenerative and scarring fibroblasts during wound healing with single-cell technologies

项目摘要 伤口成纤维细胞通过产生细胞外基质（ECM）在伤口愈合过程中发挥关键作用， 细胞再生的物理框架，补充皮肤的结构强度。当伤口成纤维细胞 促进皮肤愈合，在成年人中，这一过程与导致疤痕的变化有关，并可能导致 非功能性修复在这里，我们假设，比较伤口成纤维细胞分化的过程中， 再生和疤痕条件将确定新的分子机制，以减少 疤痕对比伤口成纤维细胞在再生和瘢痕形成过程中的分化途径， 增加我们对驱动这些过程的分子机制的理解，并可以突出新的 治疗方法的目标。在这项提案中，我们的目标是克服以前的技术 伤口成纤维细胞异质性的局限性以及通过适应和部署缺乏特异性标志物 新的单细胞基因组技术在已建立的伤口愈合模型。这种方法将使 驱动伤口成纤维细胞分化的内在和外在成分的高分辨率解构 在再生和结疤过程中转变为异质状态。首先，在目标1中，我们将采用单细胞跟踪 在我们的实验室开发的工具，细胞标记，用于部署在再生和疤痕的背景下， 体内伤口愈合。CellTagging是使用表达的病毒递送条形码对细胞进行独特标记。 作为转录物，使得能够捕获与细胞身份和功能平行的谱系信息。我们将调整我们的 CellTagging技术用于跟踪伤口再生和愈合过程中的伤口成纤维细胞来源和分化， 疤痕愈合该策略将包括标记从胚胎中回收的成纤维细胞（促进 再生）和成人（促进疤痕形成）未受伤的皮肤，然后将它们移植到宿主中， 伤人在这里，我们的目标是表征伤口成纤维细胞的异质性和起源， 未受伤皮肤中的成纤维细胞群。我们将使用我们专有的基因调控网络重建 算法，CellOracle，以确定关键的调控因素，驱动伤口成纤维细胞分化， 再生和结疤条件。在我们的补充目标2中，我们建议研究伤口成纤维细胞 与细胞微环境的相互作用。为了实现这一点，我们将执行多路复用错误鲁棒性 荧光原位杂交（MERFISH）对胚胎移植伤口切片的检测 （再生的）或成人（瘢痕形成的）成纤维细胞，以基于特异性抗体的表达来鉴定细胞群。 伤口上的标记使用已建立的计算工具，我们将分配细胞的身份和状态， 组织内的单个细胞，创建投射到伤口床组织学上的基因表达图。这 将使我们能够确定再生和瘢痕成纤维细胞的关键相互作用伙伴。在一起，成功 本文提出的目标将支持前所未有的定量伤口成纤维细胞分化和起源。 这一知识将使识别关键的分子机制，可用于促进 在伤口愈合过程中再生和减少疤痕。

PROJECT SUMMARY Wound fibroblasts play a crucial role during wound healing by producing extracellular matrix (ECM), providing a physical framework for cell repopulation, replenishing the skin's structural strength. While wound fibroblasts promote skin healing, in adults this process is associated with changes that lead to scarring and can result in non-functional repair. Here, we hypothesize that the comparison of wound fibroblast differentiation during regeneration and scarring conditions will identify novel molecular mechanisms to diminish scarring. Contrasting the differentiation paths of wound fibroblasts in regenerative and scarring processes will increase our understanding of the molecular mechanisms that drive these processes and could highlight new targets for therapeutic approaches. In this proposal, we aim to overcome previous technical limitations surrounding wound fibroblast heterogeneity and a lack of specific markers by adapting and deploying novel single-cell genomic technologies in an established wound healing model. This approach will enable the high-resolution deconstruction of the intrinsic and extrinsic components driving wound fibroblast differentiation to heterogeneous states during regeneration and scarring. First, in Aim 1, we will adapt a single-cell tracking tool developed in our laboratory, CellTagging, for deployment in the context of regenerative and scarring in vivo wound healing. CellTagging is the unique labeling of cells using virus-delivered barcodes that are expressed as transcripts, enabling capture of lineage information in parallel with cell identity and function. We will adapt our CellTagging technology to track the wound fibroblast origins and differentiation during wound regenerative and scarring healing. This strategy will consist of labeling fibroblasts recovered from embryonic (promote regeneration) and adult (promote scarring) uninjured skin, followed by their transplant into a host and subsequent wounding. Here, we aim to characterize heterogeneity and origin of wound fibroblasts tracing their lineage back to fibroblast populations in uninjured skin. We will use our proprietary gene regulatory network reconstruction algorithm, CellOracle, to identify key regulatory factors driving wound fibroblast differentiation during regeneration and scarring conditions. In our complementary Aim 2, we propose to investigate wound fibroblast interactions with their cellular microenvironment in situ. To achieve this, we will perform multiplex error-robust fluorescence in situ hybridization (MERFISH) on sections from wounds transplanted with embryonic (regenerative) or adult (scarring) fibroblasts to identify cell populations based on the expression of specific markers in the wound bed. Using established computational tools, we will assign cell identities and states to individual cells within the tissue, creating a map of gene expression projected onto wound bed histology. This will allow us to identify key interacting partners for regenerative and scarring fibroblasts. Together, the success of the aims proposed here will support unprecedented quantitation of wound fibroblast differentiation and origins. This knowledge will enable the identification of key molecular mechanisms that could be used to promote regeneration and diminish scarring during wound healing.