Molecular Programs Driving Skin Regeneration and Scarring at Single-cell Resolution
Creators: name: Sinha, Sarthak
Publisher: name: Veterinary Medicine
Year: 2022
Subjects: subject: Wound Healing
subject: Skin Regeneration
subject: Hair regeneration
subject: Fibrotic Scar
subject: Fibroblast Biology
subject: Neutrophil Heterogeneity
subject: Single-cell Transcriptomics
subject: Single-cell Epigenomics
subject: Animal Models for Wound Healing
subject: Biological Sciences
subject: Biology--Cell
subject: Biology--Molecular
subject: Biology--Bioinformatics

description: Tissue regeneration after complete amputation, where epithelial and parenchymal cells are lost, is restricted to certain phyla and tissue types. Compared to metazoans (i.e., planarians) that can regenerate new tissues, mammalian evolution has favored reparative processes that drive scar formation. Skin wound healing in adult mammals manifests the detrimental impact of these selection pressures as rapid closure happens at the expense of generating a fibrotic scar. Under some contexts, however, mammalian skin wounds elicit a regenerative response resulting in de novo formation of appendages such as hair follicles (HFs), glands and adipose. Elucidating latent mechanisms driving divergent outcomes (regeneration vs scar-formation) holds significant value as they may enable novel strategies to regenerate skin. In this three-pronged thesis, I employ single-cell genomics to decode programs that bias healing towards regenerative or fibrotic fates in distantly related mammals to provide a molecular blueprint for regenerating human skin.The first set of projects (Chapters 2 and 3) optimize molecular and computational workflows to reconstruct tissue ecosystems by measuring transcriptional and epigenetic states of individual cells. These methods provide a background for the primary research that follows.The second set of projects (Chapters 4 and 5) leverage single-cell infrastructure to ask why specific domains within large excisional wounds regenerate, whereas surrounding regions (and the entirety of small) wounds succumb to scarring. Using complementary fate-mapping strategies, we show that extrafollicular (and not pre-existing HF) fibroblasts are the primary source of dermal regeneration as they acquire inductive fates that subsequently instructs hair follicle neogenesis and differentiate to form adipocytes. This remarkable plasticity is an emergent property driven by complementary layers of regulatory control that co-opt latent morphogenic programs to drive regeneration. Specifically, our work nominates Runx1, retinoic acid, and Hic1 signalling as drivers of regenerative competence in wound-activated myofibroblasts. In Chapter 5, I hypothesize that differential signaling within local wound microenvironments may unmask this enhanced fibroblast plasticity. Unbiased assessment of epithelial and hematopoietic cells revealed neutrophils as the most discrepant cell type distinguishing regenerative and fibrotic domains. By combining mAbs targeting neutrophils at early stages of healing and single-cell profiling of wounds, we identify a pool of non-canonical immature neutrophils that repattern the immune-stromal crosstalk to increase regeneration-competent fibroblasts. Indeed, recruitment of immature neutrophils resulted in near-complete regeneration marked by a 15-fold increase in neogenic HFs. These findings highlight that functionally distinct neutrophils bias molecular events during healing and provide an impetus for uncovering neutrophil heterogeneity in human health and disease. To this end, Chapter 6 describes a set of opportunistic studies examining neutrophil response in critically ill COVID-19 patients. We show that COVID-19, compared to bacterial ARDS, is associated with an expansion of interferon- and prostaglandin-active neutrophil states. Intriguingly, immunosuppression with dexamethasone, a corticosteroid that improves outcomes in critically ill COVID-19 patients, expanded immunosuppressive immature neutrophils that were highly reminiscent of those seen during regenerative skin healing. Together, this work uncovers regulatory programs governing fibroblast functions in exquisite detail and casts a conserved pool of immature neutrophils as orchestrators of a regenerative immune milieu that augments fibroblasts’ regenerative capacity.Finally, Chapter 7 builds on immune-stromal crosstalk by asking what shapes the host inflammatory response following skin injury. We employ adult reindeer as a powerful mammalian model to study wound healing as antler velvet regenerates whereas back skin forms scar in response to an identical full-thickness injury. Single-cell multi-omics revealed that while uninjured velvet fibroblasts resembled human fetal fibroblasts, back skin fibroblasts were enriched in pro-inflammatory features resembling adult human fibroblasts. Injury elicited site-specific polarization; back skin fibroblasts amplified the immune response, whereas velvet fibroblasts adopted an immunosuppressive state leading to restrained myeloid maturation and hastened immune resolution, enabling fibroblast fate reversion and reacquisition of regenerative competence. Ectopic transplantation of velvet to back skin resulted in progressive canalization of fetal-like fibroblasts towards pro-inflammatory states which correlated with acquisition of fibrotic priming. Indeed, exogenous application of back skin fibroblast-secreted chemokines ameliorated, while neutralization of fibroblast-derived CSF1 and CXCL12 promoted, regeneration. These findings suggest fibrotic healing is driven by fibroblast-potentiated inflammatory signals that amplify effector immune programs. Together my work suggests that decoupling stromal-immune crosstalk represents an important avenue for mitigating scar.