Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

The nervous system and adipose tissues: a tale of dialogues.

White, beige, and brown adipose tissues play a crucial role in maintaining energy homeostasis. Due to the heterogeneous and diffuse nature of fat pads, this balance requires a fine and coordinated control of many actors and therefore permanent dialogues between these tissues and the central nervous system. For about two decades, many studies have been devoted to describe the neuro-anatomical and functional complexity involved to ensure this dialogue. Thus, if it is now clearly demonstrated that there is an efferent sympathetic innervation of different fat depots controlling plasticity as well as metabolic functions of the fat pad, the crucial role of sensory innervation capable of detecting local signals informing the central nervous system of the metabolic state of the relevant pads is much more recent. The purpose of this review is to provide the current state of knowledge on this subject.

Development and function of smooth muscle cells is modulated by Hic1 in mouse testis.

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (Hic1; also known as ZBTB29) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse. Postnatal deletion of Hic1 in smooth muscle cells led to their increased proliferation and resulted in dilatation of seminiferous tubules, with increased numbers of PMCs. These seminiferous tubules contained fewer Sertoli cells and more spermatogonia, and fibronectin was not detected in their basement membrane. The expression levels of genes encoding smooth muscle contractile proteins, Acta2 and Cnn1, were downregulated in the smooth muscle cells lacking Hic1, and the seminiferous tubules appeared to have reduced contractility. These data imply a role for Hic1 in determining the size of seminiferous tubules by regulating postnatal smooth muscle cell proliferation, subsequently affecting spermatogenesis in adulthood.

Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences.

Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility.

Novel AAV variants with improved tropism for human Schwann cells.

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.

Primary cilia signaling in astrocytes mediates development and regional-specific functional specification.

Astrocyte diversity is greatly influenced by local environmental modulation. Here we report that the majority of astrocytes across the mouse brain possess a singular primary cilium localized to the cell soma. Comparative single-cell transcriptomics reveals that primary cilia mediate canonical SHH signaling to modulate astrocyte subtype-specific core features in synaptic regulation, intracellular transport, energy and metabolism. Independent of canonical SHH signaling, primary cilia are important regulators of astrocyte morphology and intracellular signaling balance. Dendritic spine analysis and transcriptomics reveal that perturbation of astrocytic cilia leads to disruption of neuronal development and global intercellular connectomes in the brain. Mice with primary ciliary-deficient astrocytes show behavioral deficits in sensorimotor function, sociability, learning and memory. Our results uncover a critical role for primary cilia in transmitting local cues that drive the region-specific diversification of astrocytes within the developing brain.

Single-cell analysis reveals distinct fibroblast plasticity during tenocyte regeneration in zebrafish.

Despite their importance in tissue maintenance and repair, fibroblast diversity and plasticity remain poorly understood. Using single-cell RNA sequencing, we uncover distinct sclerotome-derived fibroblast populations in zebrafish, including progenitor-like perivascular/interstitial fibroblasts, and specialized fibroblasts such as tenocytes. To determine fibroblast plasticity in vivo, we develop a laser-induced tendon ablation and regeneration model. Lineage tracing reveals that laser-ablated tenocytes are quickly regenerated by preexisting fibroblasts. By combining single-cell clonal analysis and live imaging, we demonstrate that perivascular/interstitial fibroblasts actively migrate to the injury site, where they proliferate and give rise to new tenocytes. By contrast, perivascular fibroblast-derived pericytes or specialized fibroblasts, including tenocytes, exhibit no regenerative plasticity. Active Hedgehog (Hh) signaling is required for the proliferation of activated fibroblasts to ensure efficient tenocyte regeneration. Together, our work highlights the functional diversity of fibroblasts and establishes perivascular/interstitial fibroblasts as tenocyte progenitors that promote tendon regeneration in a Hh signaling-dependent manner.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19.

Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.

Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration.

The wound healing response is one of most primitive and conserved physiological responses in the animal kingdom, as restoring tissue integrity/homeostasis can be the difference between life and death. Wound healing in mammals is mediated by immune cells and inflammatory signaling molecules that regulate tissue resident cells, including local progenitor cells, to mediate closure of the wound through formation of a scar. Proteoglycan 4 (PRG4), a protein found throughout the animal kingdom from fish to elephants, is best known as a glycoprotein that reduces friction between articulating surfaces (e.g. cartilage). Previously, PRG4 was also shown to regulate the inflammatory and fibrotic response. Based on this, we asked whether PRG4 plays a role in the wound healing response. Using an ear wound model, topical application of exogenous recombinant human (rh)PRG4 hastened wound closure and enhanced tissue regeneration. Our results also suggest that rhPRG4 may impact the fibrotic response, angiogenesis/blood flow to the injury site, macrophage inflammatory dynamics, recruitment of immune and increased proliferation of adult mesenchymal progenitor cells (MPCs) and promoting chondrogenic differentiation of MPCs to form the auricular cartilage scaffold of the injured ear. These results suggest that PRG4 has the potential to suppress scar formation while enhancing connective tissue regeneration post-injury by modulating aspects of each wound healing stage (blood clotting, inflammation, tissue generation and tissue remodeling). Therefore, we propose that rhPRG4 may represent a potential therapy to mitigate scar and improve wound healing.

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia.

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell-deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.

Pain sensing neurons promote tissue regeneration in adult mice.

Tissue repair after injury in adult mammals, usually results in scarring and loss of function in contrast to lower vertebrates such as the newt and zebrafish that regenerate. Understanding the regulatory processes that guide the outcome of tissue repair is therefore a concerning challenge for regenerative medicine. In multiple regenerative animal species, the nerve dependence of regeneration is well established, but the nature of the innervation required for tissue regeneration remains largely undefined. Using our model of induced adipose tissue regeneration in adult mice, we demonstrate here that nociceptive nerves promote regeneration and their removal impairs tissue regeneration. We also show that blocking the receptor for the nociceptive neuropeptide calcitonin gene-related peptide (CGRP) inhibits regeneration, whereas CGRP administration induces regeneration. These findings reveal that peptidergic nociceptive neurons are required for adult mice tissue regeneration.

Driving regeneration, instead of healing, in adult mammals: the decisive role of resident macrophages through efferocytosis.

Tissue repair after lesion usually leads to scar healing and thus loss of function in adult mammals. In contrast, other adult vertebrates such as amphibians have the ability to regenerate and restore tissue homeostasis after lesion. Understanding the control of the repair outcome is thus a concerning challenge for regenerative medicine. We recently developed a model of induced tissue regeneration in adult mice allowing the comparison of the early steps of regenerative and scar healing processes. By using studies of gain and loss of function, specific cell depletion approaches, and hematopoietic chimeras we demonstrate here that tissue regeneration in adult mammals depends on an early and transient peak of granulocyte producing reactive oxygen species and an efficient efferocytosis specifically by tissue-resident macrophages. These findings highlight key and early cellular pathways able to drive tissue repair towards regeneration in adult mammals.

Transcriptional Profiling of the Adult Hair Follicle Mesenchyme Reveals R-spondin as a Novel Regulator of Dermal Progenitor Function.

The adult hair follicle (HF) undergoes successive regeneration driven by resident epithelial stem cells and neighboring mesenchyme. Recent work described the existence of HF dermal stem cells (hfDSCs), but the genetic regulation of hfDSCs and their daughter cell lineages in HF regeneration remains unknown. Here we prospectively isolate functionally distinct mesenchymal compartment in the HF (dermal cup [DC; includes hfDSCs] and dermal papilla) and define the transcriptional programs involved in hfDSC function and acquisition of divergent mesenchymal fates. From this, we demonstrate cross-compartment mesenchymal signaling within the HF niche, whereby DP-derived R-spondins act to stimulate proliferation of both hfDSCs and epithelial progenitors during HF regeneration. Our findings describe unique transcriptional programs that underlie the functional heterogeneity among specialized fibroblasts within the adult HF and identify a novel regulator of mesenchymal progenitor function during tissue regeneration.

Dysfunction of Hair Follicle Mesenchymal Progenitors Contributes to Age-Associated Hair Loss.

Skin aging is accompanied by hair loss due to impairments in hair follicle (HF) epithelial progenitor cells and their mesenchymal niche. This inductive mesenchyme, called dermal papilla (DP), undergoes progressive cell loss and eventual miniaturization that contributes to HF pathogenesis. Using laser ablation and fate mapping, we show that HF dermal stem cells (hfDSCs) reconstitute the damaged DP and maintain hair growth, suggesting that hfDSC dysfunction may trigger degeneration of the inductive niche. Fate mapping over 24 months revealed progressive hfDSC depletion, and in vivo clonal analysis of aged hfDSCs showed impaired self-renewal and biased differentiation. Single-cell RNA-seq confirmed hfDSCs as a central precursor, giving rise to divergent mesenchymal trajectories. In aged skin, hfDSCs exhibited senescent-like characteristics, and senescence-associated secretory phenotypes were identified in the aging HF mesenchyme. These results clarify fibroblast dynamics within the HF and suggest that progressive dysfunction within the mesenchymal progenitor pool contributes to age-related hair loss.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.