Stimulation of skeletal stem cells in the growth plate promotes linear bone growth.

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.

Tenascin-C expressing touch dome keratinocytes exhibit characteristics of all epidermal lineages.

The touch dome (TD) keratinocytes are specialized epidermal cells that intimately associate with the light touch sensing Merkel cells (MCs). The TD keratinocytes function as a niche for the MCs and can induce de novo hair follicles upon stimulation; however, how the TD keratinocytes are maintained during homeostasis remains unclear. scRNA-seq identified a specific TD keratinocyte marker, Tenascin-C (TNC). Lineage tracing of Tnc-expressing TD keratinocytes revealed that these cells maintain themselves as an autonomous epidermal compartment and give rise to MCs upon injury. Molecular characterization uncovered that, while the transcriptional and chromatin landscape of the TD keratinocytes is remarkably similar to that of the interfollicular epidermal keratinocytes, it also shares certain molecular signatures with the hair follicle keratinocytes. Our study highlights that the TD keratinocytes in the adult skin have molecular characteristics of keratinocytes of diverse epidermal lineages.

A Roadmap for a Consensus Human Skin Cell Atlas and Single-Cell Data Standardization.

Single-cell technologies have become essential to driving discovery in both basic and translational investigative dermatology. Despite the multitude of available datasets, a central reference atlas of normal human skin, which can serve as a reference resource for skin cell types, cell states, and their molecular signatures, is still lacking. For any such atlas to receive broad acceptance, participation by many investigators during atlas construction is an essential prerequisite. As part of the Human Cell Atlas project, we have assembled a Skin Biological Network to build a consensus Human Skin Cell Atlas and outline a roadmap toward that goal. We define the drivers of skin diversity to be considered when selecting sequencing datasets for the atlas and list practical hurdles during skin sampling that can result in data gaps and impede comprehensive representation and technical considerations for tissue processing and computational analysis, the accounting for which should minimize biases in cell type enrichments and exclusions and decrease batch effects. By outlining our goals for Atlas 1.0, we discuss how it will uncover new aspects of skin biology.

Autophagy critically controls skin inflammation and apoptosis-induced stem cell activation.

Macroautophagy/autophagy is a cellular recycling program regulating cell survival and controlling inflammatory responses in a context-dependent manner. Here, we demonstrate that keratinocyte-selective ablation of Atg16l1, an essential autophagy mediator, results in exacerbated inflammatory and neoplastic skin responses. In addition, mice lacking keratinocyte autophagy exhibit precocious onset of hair follicle growth, indicating altered activation kinetics of hair follicle stem cells (HFSCs). These HFSCs also exhibit expanded potencies in an autophagy-deficient context as shown by de novo hair follicle formation and improved healing of abrasion wounds. ATG16L1-deficient keratinocytes are markedly sensitized to apoptosis. Compound deletion of RIPK3-dependent necroptotic and CASP8-dependent apoptotic responses or of TNFRSF1A/TNFR1 reveals that the enhanced sensitivity of autophagy-deficient keratinocytes to TNF-dependent cell death is driving altered activation of HFSCs. Together, our data demonstrate that keratinocyte autophagy dampens skin inflammation and tumorigenesis but curtails HFSC activation by restraining apoptotic responses.Abbreviations: ATG16L1: autophagy related 16 like 1; DMBA: 2,4-dimethoxybenzaldehyde; DP: dermal papilla; EpdSCs: epidermal stem cells; Gas6: growth arrest specific 6; HF: hair follicle; HFSC: hair follicle stem cell; IFE: interfollicular epidermis; KRT5: keratin 5; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PMK: primary mouse keratinocyte; RIPK3: receptor-interacting serine-threonine kinase 3; scRNAseq: single-cell RNA-sequencing; SG: sebaceous gland; TEWL: transepidermal water loss; TPA: 12-O-tetradecanoylphorbol-13-acetate; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a; UMAP: uniform manifold approximation and projection.

Molecular and spatial landmarks of early mouse skin development.

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.

Skin mesenchymal niches maintain and protect AML-initiating stem cells.

Leukemia cutis or leukemic cell infiltration in skin is one of the common extramedullary manifestations of acute myeloid leukemia (AML) and signifies a poorer prognosis. However, its pathogenesis and maintenance remain understudied. Here, we report massive AML cell infiltration in the skin in a transplantation-induced MLL-AF9 AML mouse model. These AML cells could regenerate AML after transplantation. Prospective niche characterization revealed that skin harbored mesenchymal progenitor cells (MPCs) with a similar phenotype as BM mesenchymal stem cells. These skin MPCs protected AML-initiating stem cells (LSCs) from chemotherapy in vitro partially via mitochondrial transfer. Furthermore, Lama4 deletion in skin MPCs promoted AML LSC proliferation and chemoresistance. Importantly, more chemoresistant AML LSCs appeared to be retained in Lama4-/- mouse skin after cytarabine treatment. Our study reveals the characteristics and previously unrecognized roles of skin mesenchymal niches in maintaining and protecting AML LSCs during chemotherapy, meriting future exploration of their impact on AML relapse.

Injury prevents Ras mutant cell expansion in mosaic skin.

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

Metabolic rewiring controlled by c-Fos governs cartilage integrity in osteoarthritis.

OBJECTIVES: The activator protein-1 (AP-1) transcription factor component c-Fos regulates chondrocyte proliferation and differentiation, but its involvement in osteoarthritis (OA) has not been functionally assessed. METHODS: c-Fos expression was evaluated by immunohistochemistry on articular cartilage sections from patients with OA and mice subjected to the destabilisation of the medial meniscus (DMM) model of OA. Cartilage-specific c-Fos knockout (c-FosΔCh) mice were generated by crossing c-fosfl/fl to Col2a1-CreERT mice. Articular cartilage was evaluated by histology, immunohistochemistry, RNA sequencing (RNA-seq), quantitative reverse transcription PCR (qRT-PCR) and in situ metabolic enzyme assays. The effect of dichloroacetic acid (DCA), an inhibitor of pyruvate dehydrogenase kinase (Pdk), was assessed in c-FosΔCh mice subjected to DMM. RESULTS: FOS-positive chondrocytes were increased in human and murine OA cartilage during disease progression. Compared with c-FosWT mice, c-FosΔCh mice exhibited exacerbated DMM-induced cartilage destruction. Chondrocytes lacking c-Fos proliferate less, have shorter collagen fibres and reduced cartilage matrix. Comparative RNA-seq revealed a prominent anaerobic glycolysis gene expression signature. Consistently decreased pyruvate dehydrogenase (Pdh) and elevated lactate dehydrogenase (Ldh) enzymatic activities were measured in situ, which are likely due to higher expression of hypoxia-inducible factor-1α, Ldha, and Pdk1 in chondrocytes. In vivo treatment of c-FosΔCh mice with DCA restored Pdh/Ldh activity, chondrocyte proliferation, collagen biosynthesis and decreased cartilage damage after DMM, thereby reverting the deleterious effects of c-Fos inactivation. CONCLUSIONS: c-Fos modulates cellular bioenergetics in chondrocytes by balancing pyruvate flux between anaerobic glycolysis and the tricarboxylic acid cycle in response to OA signals. We identify a novel metabolic adaptation of chondrocytes controlled by c-Fos-containing AP-1 dimers that could be therapeutically relevant.

Cell cycle controls long-range calcium signaling in the regenerating epidermis.

Skin homeostasis is maintained by stem cells, which must communicate to balance their regenerative behaviors. Yet, how adult stem cells signal across regenerative tissue remains unknown due to challenges in studying signaling dynamics in live mice. We combined live imaging in the mouse basal stem cell layer with machine learning tools to analyze patterns of Ca2+ signaling. We show that basal cells display dynamic intercellular Ca2+ signaling among local neighborhoods. We find that these Ca2+ signals are coordinated across thousands of cells and that this coordination is an emergent property of the stem cell layer. We demonstrate that G2 cells are required to initiate normal levels of Ca2+ signaling, while connexin43 connects basal cells to orchestrate tissue-wide coordination of Ca2+ signaling. Lastly, we find that Ca2+ signaling drives cell cycle progression, revealing a communication feedback loop. This work provides resolution into how stem cells at different cell cycle stages coordinate tissue-wide signaling during epidermal regeneration.

ARF suppression by MYC but not MYCN confers increased malignancy of aggressive pediatric brain tumors.

Medulloblastoma, the most common malignant pediatric brain tumor, often harbors MYC amplifications. Compared to high-grade gliomas, MYC-amplified medulloblastomas often show increased photoreceptor activity and arise in the presence of a functional ARF/p53 suppressor pathway. Here, we generate an immunocompetent transgenic mouse model with regulatable MYC that develop clonal tumors that molecularly resemble photoreceptor-positive Group 3 medulloblastoma. Compared to MYCN-expressing brain tumors driven from the same promoter, pronounced ARF silencing is present in our MYC-expressing model and in human medulloblastoma. While partial Arf suppression causes increased malignancy in MYCN-expressing tumors, complete Arf depletion promotes photoreceptor-negative high-grade glioma formation. Computational models and clinical data further identify drugs targeting MYC-driven tumors with a suppressed but functional ARF pathway. We show that the HSP90 inhibitor, Onalespib, significantly targets MYC-driven but not MYCN-driven tumors in an ARF-dependent manner. The treatment increases cell death in synergy with cisplatin and demonstrates potential for targeting MYC-driven medulloblastoma.

Plasticity of Epithelial Cells during Skin Wound Healing.

Epithelial tissues line the outer surfaces of the mammalian body and protect from external harm. In skin, the epithelium is maintained by distinct stem cell populations residing in the interfollicular epidermis and various niches of the hair follicle. These stem cells give rise to the stratified epidermal layers and the protective hair coat, while being confined to their respective niches. Upon injury, however, all stem cell progenies can leave their niche and collectively contribute to a central wound healing process, called reepithelialization, for restoring the skin's barrier function. This review explores how epithelial cells from distinct niches respond and adapt during acute wound repair. We discuss when and where cells sense and react to damage, how cellular identity is regulated at the molecular and behavioral level, and how cells memorize past experiences and their origin. This collective knowledge highlights cellular plasticity as a brilliant feature of epithelial tissues to heal.

Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1-4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

Retrograde movements determine effective stem cell numbers in the intestine.

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance.

Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.

Diagnostic Accuracy of Point-of-Care Fluorescence Imaging for the Detection of Bacterial Burden in Wounds: Results from the 350-Patient Fluorescence Imaging Assessment and Guidance Trial.

Objective: High bacterial load contributes to chronicity of wounds and is diagnosed based on assessment of clinical signs and symptoms (CSS) of infection, but these characteristics are poor predictors of bacterial burden. Point-of-care fluorescence imaging (FL) MolecuLight i:X can improve identification of wounds with high bacterial burden (>104 colony-forming unit [CFU]/g). FL detects bacteria, whether planktonic or in biofilm, but does not distinguish between the two. In this study, diagnostic accuracy of FL was compared to CSS during routine wound assessment. Postassessment, clinicians were surveyed to assess impact of FL on treatment plan. Approach: A prospective multicenter controlled study was conducted by 20 study clinicians from 14 outpatient advanced wound care centers across the United States. Wounds underwent assessment for CSS followed by FL. Biopsies were collected to confirm total bacterial load. Three hundred fifty patients completed the study (138 diabetic foot ulcers, 106 venous leg ulcers, 60 surgical sites, 22 pressure ulcers, and 24 others). Results: Around 287/350 wounds (82%) had bacterial loads >104 CFU/g, and CSS missed detection of 85% of these wounds. FL significantly increased detection of bacteria (>104 CFU/g) by fourfold, and this was consistent across wound types (p < 0.001). Specificity of CSS+FL remained comparably high to CSS (p = 1.0). FL information modified treatment plans (69% of wounds), influenced wound bed preparation (85%), and improved overall patient care (90%) as reported by study clinicians. Innovation: This novel noncontact, handheld FL device provides immediate, objective information on presence, location, and load of bacteria at point of care. Conclusion: Use of FL facilitates adherence to clinical guidelines recommending prompt detection and removal of bacterial burden to reduce wound infection and facilitate healing.

Epiphyseal Cartilage Formation Involves Differential Dynamics of Various Cellular Populations During Embryogenesis.

A joint connects two or more bones together to form a functional unit that allows different types of bending and movement. Little is known about how the opposing ends of the connected bones are developed. Here, applying various lineage tracing strategies we demonstrate that progenies of Gdf5-, Col2-, Prrx1-, and Gli1-positive cells contribute to the growing epiphyseal cartilage in a spatially asymmetrical manner. In addition, we reveal that cells in the cartilaginous anlagen are likely to be the major sources for epiphyseal cartilage. Moreover, Gli1-positive cells are found to proliferate along the skeletal edges toward the periarticular region of epiphyseal surface. Finally, a switch in the mechanism of growth from cell division to cell influx likely occurs in the epiphyseal cartilage when joint cavitation has completed. Altogether, our findings reveal an asymmetrical mechanism of growth that drives the formation of epiphyseal cartilage ends, which might implicate on how the articular surface of these skeletal elements acquires their unique and sophisticated shape during embryonic development.

Coordinated hedgehog signaling induces new hair follicles in adult skin.

Hair follicle (HF) development is orchestrated by coordinated signals from adjacent epithelial and mesenchymal cells. In humans this process only occurs during embryogenesis and viable strategies to induce new HFs in adult skin are lacking. Here, we reveal that activation of Hedgehog (Hh) signaling in adjacent epithelial and stromal cells induces new HFs in adult, unwounded dorsal mouse skin. Formation of de novo HFs recapitulated embryonic HF development, and mature follicles produced hair co-occurring with epithelial tumors. In contrast, Hh-pathway activation in epithelial or stromal cells alone resulted in tumor formation or stromal cell condensation respectively, without induction of new HFs. Provocatively, adjacent epithelial-stromal Hh-pathway activation induced de novo HFs also in hairless paw skin, divorced from confounding effects of pre-existing niche signals in haired skin. Altogether, cell-type-specific modulation of a single pathway is sufficient to reactivate embryonic programs in adult tissues, thereby inducing complex epithelial structures even without wounding.

We are born with all the hair follicles that we will ever have in our life. These structures are maintained by different types of cells (such as keratinocytes and fibroblasts) that work together to create hair. Follicles form in the embryo thanks to complex molecular signals, which include a molecular cascade known as the Hedgehog signaling pathway. After birth however, these molecular signals are shut down to avoid conflicting messages ­ inappropriate activation of Hedgehog signaling in adult skin, for instance, leads to tumors. This means that our skin loses the ability to make new hair follicles, and if skin is severely damaged it cannot regrow hair or produce the associated sebaceous glands that keep skin moisturized. Being able to create new hair follicles in adult skin would be both functionally and aesthetically beneficial for patients in need, for example, burn victims. Overall, it would also help to understand if and how it is possible to reactivate developmental programs after birth. To investigate this question, Sun, Are et al. triggered Hedgehog signaling in the skin cells of genetically modified mice; this was done either in keratinocytes, in fibroblasts, or in both types of cells. The experiments showed that Hedgehog signaling could produce new hair follicles, but only when activated in keratinocytes and fibroblasts together. The process took several weeks, mirrored normal hair follicle development and resulted in new hair shafts. The follicles grew on both the back of mice, where hair normally occurs, and even in paw areas that are usually hairless. Not unexpectedly the new hair follicles were accompanied with skin tumors. But, promisingly, treatment with Hedgehog-pathway inhibitor Vismodegib restricted tumor growth while keeping the new follicles intact. This suggests that future work on improving "when and where" Hedgehog signaling is activated may allow the formation of new follicles in adult skin with fewer adverse effects.

Cruciate ligament, patellar tendon, and patella formation involves differential cellular sources and dynamics as joint cavitation proceeds.

BACKGROUND: Cruciate ligament (CL) and patellar tendon (PT) are important elements of the knee joint, uniting femur, patella, and tibia into a single functional unit. So far, knowledge on the developmental mechanism of CL, PT, and patella falls far behind other skeletal tissues. RESULTS: Here, employing various lineage tracing strategies we investigate the cellular sources and dynamics that drive CL, PT, and patella formation during mouse embryonic development. We show that Gdf5 and Gli1 are generally expressed in the same cell population that only contributes to CL, but not PT or patella development. In addition, Col2 is expressed in two independent cell populations before and after joint cavitation, where the former contributes to the CL and the dorsal part of the PT and the latter contributes to the patella. Moreover, Prrx1 is always expressed in CL and PT progenitors, but not patella progenitors where it is switched off after joint cavitation. Finally, we reveal that patella development employs different cellular dynamics before and after joint cavitation. CONCLUSIONS: Our findings delineate the expression changes of several skeletogenesis-related genes before and after joint cavitation, and provide an indication on the cellular dynamics underlying ligament, tendon, and sesamoid bone formation during embryogenesis.