A transit-amplifying progenitor with biphasic behavior contributes to epidermal renewal.

Transit-amplifying (TA) cells are progenitors that undergo an amplification phase followed by transition into an extinction phase. A long postulated epidermal TA progenitor with biphasic behavior has not yet been experimentally observed in vivo. Here, we identify such a TA population using clonal analysis of Aspm-CreER genetic cell-marking in mice, which uncovers contribution to both homeostasis and injury repair of adult skin. This TA population is more frequently dividing than a Dlx1-CreER-marked long-term self-renewing (e.g. stem cell) population. Newly developed generalized birth-death modeling of long-term lineage tracing data shows that both TA progenitors and stem cells display neutral competition, but only the stem cells display neutral drift. The quantitative evolution of a nascent TA cell and its direct descendants shows that TA progenitors indeed amplify the basal layer before transition and that the homeostatic TA population is mostly in extinction phase. This model will be broadly useful for analyzing progenitors whose behavior changes with their clone age. This work identifies a long-missing class of non-self-renewing biphasic epidermal TA progenitors and has broad implications for understanding tissue renewal mechanisms.

Alk1 acts in non-endothelial VE-cadherin+ perineurial cells to maintain nerve branching during hair homeostasis.

Vascular endothelial (VE)-cadherin is a well-recognized endothelial cell marker. One of its interacting partners, the TGF-ß receptor Alk1, is essential in endothelial cells for adult skin vasculature remodeling during hair homeostasis. Using single-cell transcriptomics, lineage tracing and gene targeting in mice, we characterize the cellular and molecular dynamics of skin VE-cadherin+ cells during hair homeostasis. We describe dynamic changes of VE-cadherin+ endothelial cells specific to blood and lymphatic vessels and uncover an atypical VE-cadherin+ cell population. The latter is not a predicted adult endovascular progenitor, but rather a non-endothelial mesenchymal perineurial cell type, which forms nerve encapsulating tubular structures that undergo remodeling during hair homeostasis. Alk1 acts in the VE-cadherin+ perineurial cells to maintain proper homeostatic nerve branching by enforcing basement membrane and extracellular matrix molecular signatures. Our work implicates the VE-cadherin/Alk1 duo, classically known as endothelial-vascular specific, in perineurial-nerve homeostasis. This has broad implications in vascular and nerve disease.

Single-cell transcriptomics of adult skin VE-cadherin expressing lineages during hair cycle.

Adult skin homeostasis involves global reorganization of dermal lineages at different stages of the mouse hair growth cycle. Vascular endothelial cadherin (VE-cadherin encoded by Cdh5 ) expressing cells from blood and lymphatic vasculature structures are known to remodel during the adult hair cycle. Here we employ single-cell RNA-sequencing (scRNA-seq) 10x-genomics analysis of FACS-sorted VE-cadherin expressing cells marked via Cdh5-CreER genetic labeling at resting (telogen) and growth (anagen) stage of hair cycle. Our comparative analysis between the two stages uncovers a persistent Ki67 + proliferative EC population and documents changes in EC population distribution and gene expression. Global gene expression changes in all the analyzed populations revealed bioenergetic metabolic changes that may drive vascular remodeling during HF growth phase, alongside a few highly restricted cluster-specific gene expression differences. This study uncovers active cellular and molecular dynamics of adult skin endothelial lineages during hair cycle that may have broad implications in adult tissue regeneration and for understanding vascular disease.

Blood endothelial ALK1-BMP4 signaling axis regulates adult hair follicle stem cell activation.

Blood vessels can play dual roles in tissue growth by transporting gases and nutrients and by regulating tissue stem cell activity via signaling. Correlative evidence implicates skin endothelial cells (ECs) as signaling niches of hair follicle stem cells (HFSCs), but functional demonstration from gene depletion of signaling molecules in ECs is missing to date. Here, we show that depletion of the vasculature-factor Alk1 increases BMP4 secretion from ECs, which delays HFSC activation. Furthermore, while previous evidence suggests a lymphatic vessel role in adult HFSC activation possibly through tissue drainage, a blood vessel role has not yet been addressed. Genetic perturbation of the ALK1-BMP4 axis in all ECs or the lymphatic ECs specifically unveils inhibition of HFSC activation by blood vessels. Our work suggests a broader relevance of blood vessels, adding adult HFSCs to the EC functional repertoire as signaling niches for the adult stem cells.

Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Hair follicle stem cells as a skin-organizing signaling center during adult homeostasis.

Stem cells are the essential source of building blocks for tissue homeostasis and regeneration. Their behavior is dictated by both cell-intrinsic cues and extrinsic cues from the microenvironment, known as the stem cell niche. Interestingly, recent work began to demonstrate that hair follicle stem cells (HFSCs) are not only passive recipients of signals from the surroundings, but also actively send out signals to modulate the organization and function of their own niches. Here, we discuss recent findings, and briefly refer to the old, on the interaction of HFSCs and their niches with the emphasis on the outwards signals from HFSCs toward their niches. We also highlight recent technology advancements that further promote our understanding of HFSC niches. Taken together, the HFSCs emerge as a skin-organizing center rich in signaling output for niche remodeling during various stages of adult skin homeostasis. The intricate crosstalk between HFSCs and their niches adds important insight to skin biology that will inform clinical and bioengineering fields aiming to build complete and functional 3D organotypic cultures for skin replacement therapies.

High-resolution single-cell transcriptomics reveals heterogeneity of self-renewing hair follicle stem cells.

Multipotent bulge stem cells (SCs) fuel the hair follicle (HF) cyclic growth during adult skin homeostasis, but their intrinsic molecular heterogeneity is not well understood. These hair follicle stem cells (HFSCs) engage in bouts of self-renewal, migration and differentiation during the hair cycle. Here, we perform high-resolution single-cell RNA sequencing (scRNA-seq) of HFSCs sorted as CD34+ /K14-H2BGFP+ from mouse skin at mid-anagen, the self-renewal stage. We dissect the transcriptomic profiles and unravel population-specific transcriptional heterogeneity. Unsupervised clustering reveals five major HFSC populations, which distinguished by known markers associated with both the bulge and the outer root sheath (ORS) underneath. These populations include quiescent bulge, ORS cellular states and proliferative cells. Lineage trajectory analysis predicted the prospective differentiation path of these cellular states and their corresponding self-renewing subpopulations. The bulge population itself can be further sub-divided into distinct subpopulations that can be mapped to the upper, mid and lower bulge regions, and present a decreasing quiescence score. Gene set enrichment analysis (GSEA) revealed new markers and suggested potentially distinct functions of the ORS and bulge subpopulations. This included communications between the upper bulge subpopulation and sensory nerves and between the upper ORS and skin vasculature, as well as enrichment of a bulge subset in cell migratory functions. The lower ORS enriched genes may potentially enable nutrients passing from the surrounding fat and vasculature cells towards the proliferating hair matrix cells. Thus, we provide a comprehensive account of HFSC molecular heterogeneity during their self-renewing stage, which enables future HF functional studies.

Stem cell-intrinsic mechanisms regulating adult hair follicle homeostasis.

Adult hair follicle stem cells (HFSCs) undergo dynamic and periodic molecular changes in their cellular states throughout the hair homeostatic cycle. These states are tightly regulated by cell-intrinsic mechanisms and by extrinsic signals from the microenvironment. HFSCs are essential not only for fuelling hair growth, but also for skin wound healing. Increasing evidence suggests an important role of HFSCs in organizing multiple skin components around the hair follicle, thus functioning as an organizing centre during adult skin homeostasis. Here, we focus on recent findings on cell-intrinsic mechanisms of HFSC homeostasis, which include transcription factors, histone modifications, DNA regulatory elements, non-coding RNAs, cell metabolism, cell polarity and post-transcriptional mRNA processing. Several transcription factors are now known to participate in well-known signalling pathways that control hair follicle homeostasis, as well as in super-enhancer activities to modulate HFSC and progenitor lineage progression. Interestingly, HFSCs have been shown to secrete molecules that are important in guiding the organization of several skin components around the hair follicle, including nerves, arrector pili muscle and vasculature. Finally, we discuss recent technological advances in the field such as single-cell RNA sequencing and live imaging, which revealed HFSC and progenitor heterogeneity and brought new light to understanding crosstalking between HFSCs and the microenvironment. The field is well on its way to generate a comprehensive map of molecular interactions that should serve as a solid theoretical platform for application in hair and skin disease and ageing.

Histone H3 K4/9/27 Trimethylation Levels Affect Wound Healing and Stem Cell Dynamics in Adult Skin.

Epigenetic mechanisms controlling adult mammalian stem cell (SC) dynamics might be critical for tissue regeneration but are poorly understood. Mouse skin and hair follicle SCs (HFSCs) display reduced histone H3 K4me3, K9me3, and K27me3 methylation levels (hypomethylation) preceding hair growth. Chemical inhibition of relevant histone demethylases impairs subsequent differentiation and growth of HFs and delays wound healing. In wounding, this impairs epithelial cell differentiation and blood vessel recruitment, but not proliferation and fibroblast recruitment. With Aspm-CreER as a newfound inter-follicular epidermis lineage-labeling tool, and Lgr5-CreER for hair follicles, we demonstrate a reduced contribution of both lineages to wound healing after interfering with hypomethylation. Blocked hypomethylation increases BMP4 expression and selectively upregulates H3 K4me3 on the Bmp4 promoter, which may explain the effects on HFSC quiescence, hair cycle, and injury repair. Thus, transient hypomethylation of histone H3 K4/9/27me3 is essential for adult skin epithelial SC dynamics for proper tissue homeostasis and repair.

Skin vasculature and hair follicle cross-talking associated with stem cell activation and tissue homeostasis.

Skin vasculature cross-talking with hair follicle stem cells (HFSCs) is poorly understood. Skin vasculature undergoes dramatic remodeling during adult mouse hair cycle. Specifically, a horizontal plexus under the secondary hair germ (HPuHG) transiently neighbors the HFSC activation zone during the quiescence phase (telogen). Increased density of HPuHG can be induced by reciprocal mutations in the epithelium (Runx1) and endothelium (Alk1) in adult mice, and is accompanied by prolonged HFSC quiescence and by delayed entry and progression into the hair growth phase (anagen). Suggestively, skin vasculature produces BMP4, a well-established HFSC quiescence-inducing factor, thus contributing to a proliferation-inhibitory environment near the HFSC. Conversely, the HFSC activator Runx1 regulates secreted proteins with previously demonstrated roles in vasculature remodeling. We suggest a working model in which coordinated remodeling and molecular cross-talking of the adult epithelial and endothelial skin compartments modulate timing of HFSC activation from quiescence for proper tissue homeostasis of adult skin.

Epigenetic control in skin development, homeostasis and injury repair.

Cell-type- and cell-state-specific patterns of covalent modifications on DNA and histone tails form global epigenetic profiles that enable spatiotemporal regulation of gene expression. These epigenetic profiles arise from coordinated activities of transcription factors and epigenetic modifiers, which result in cell-type-specific outputs in response to dynamic environmental conditions and signalling pathways. Recent mouse genetic and functional studies have highlighted the physiological significance of global DNA and histone epigenetic modifications in skin. Importantly, specific epigenetic profiles are emerging for adult skin stem cells that are associated with their cell fate plasticity and proper activity in tissue regeneration. We can now begin to draw a more comprehensive picture of how epigenetic modifiers orchestrate their cell-intrinsic role with microenvironmental cues for proper skin development, homeostasis and wound repair. The field is ripe to begin to implement these findings from the laboratory into skin therapies.

Runx1 Role in Epithelial and Cancer Cell Proliferation Implicates Lipid Metabolism and Scd1 and Soat1 Activity.

The role of lipid metabolism in epithelial stem cell (SC) function and carcinogenesis is poorly understood. The transcription factor Runx1 is known to regulate proliferation in mouse epithelial hair follicle (HF) SCs in vivo and in several mouse and human epithelial cancers. We found a novel subset of in vivo Runx1 HFSC target genes related to lipid metabolism and demonstrated changes in distinct classes of lipids driven by Runx1. Inhibition of lipid-enzymes Scd1 and Soat1 activity synergistically reduces proliferation of mouse skin epithelial cells and of human skin and oral squamous cell carcinoma cultured lines. Varying Runx1 levels induces changes in skin monounsaturated fatty acids (e.g., oleate, a product of Scd1) as shown by our lipidome analysis. Furthermore, varying Runx1 levels, the inhibition of Scd1, or the addition of Scd1-product oleate, individually affects the plasma membrane organization (or fluidity) in mouse keratinocytes. These factors also affect the strength of signal transduction through the membranes for Wnt, a pathway that promotes epithelial (cancer) cell proliferation and HFSC activation. Our working model is that HFSC factor Runx1 modulates the fatty acid production, which affects membrane organization, facilitating signal transduction for rapid proliferation of normal and cancer epithelial cells. Stem Cells 2018;36:1603-1616.

Linking chromatin dynamics, cell fate plasticity, and tissue homeostasis in adult mouse hair follicle stem cells.

Cellular plasticity for fate acquisition is associated with distinct chromatin states, which include histone modifications, dynamic association of chromatin factors with the DNA, and global chromatin compaction and nuclear organization. While embryonic stem cell (ESC) plasticity in vitro and its link with chromatin states have been characterized in depth, little is known about tissue stem cell plasticity in vivo, during adult tissue homeostasis. Recently, we reported a distinct globally low level of histone H3 K4/9/27me3 in mouse hair follicle stem cells (HFSCs) during quiescence. This occurred at the stage preceding fate acquisition, when HFSC fate plasticity must be at its highest. This hypomethylated state was required for proper skin homeostasis and timely hair cycle. Here, we show both in the live tissue and in cell culture that at quiescence HFSCs have higher exchange rates for core histone H2B when compared with proliferative or differentiated cells. This denoted a hyperdynamic chromatin state, which was previously associated with high cell fate plasticity in ESCs. Moreover, we find that quiescent HFSCs display a higher propensity for de-differentiation in response to Yamanaka's reprogramming factors in vivo. These results further support our recent model in which HFSCs render their chromatin into a specific state at quiescence, which is attuned to higher cell fate plasticity.

Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation.

Cell proliferation is essential to rapid tissue growth and repair, but can result in replication-associated genome damage. Here, we implicate the transcription factor Gata6 in adult mouse hair follicle regeneration where it controls the renewal of rapidly proliferating epithelial (matrix) progenitors and hence the extent of production of terminally differentiated lineages. We find that Gata6 protects against DNA damage associated with proliferation, thus preventing cell cycle arrest and apoptosis. Furthermore, we show that in vivo Gata6 stimulates EDA-receptor signaling adaptor Edaradd level and NF-κB pathway activation, known to be important for DNA damage repair and stress response in general and for hair follicle growth in particular. In cultured keratinocytes, Edaradd rescues DNA damage, cell survival, and proliferation of Gata6 knockout cells and restores MCM10 expression. Our data add to recent evidence in embryonic stem and neural progenitor cells, suggesting a model whereby developmentally regulated transcription factors protect from DNA damage associated with proliferation at key stages of rapid tissue growth. Our data may add to understanding why Gata6 is a frequent target of amplification in cancers.

Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.

The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1(CreER) and Slc1a3(CreER) genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other's territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

Signalling couples hair follicle stem cell quiescence with reduced histone H3 K4/K9/K27me3 for proper tissue homeostasis.

Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

A 3D Porous Gelatin-Alginate-Based-IPN Acts as an Efficient Promoter of Chondrogenesis from Human Adipose-Derived Stem Cells.

Cartilage has limited regeneration potential. Thus, there is an imperative need to develop new strategies for cartilage tissue engineering (CTE) amenable for clinical use. Recent CTE approaches rely on optimal cell-scaffold interactions, which require a great deal of optimization. In this study we attempt to build a novel gelatin- (G-) alginate- (A-) polyacrylamide (PAA) 3D interpenetrating network (IPN) with superior performance in promoting chondrogenesis from human adipose-derived stem cells (hADSCs). We show that our G-A-PAA scaffold is capable of supporting hADSCs proliferation and survival, with no apparent cytotoxic effect. Moreover, we find that after exposure to prochondrogenic conditions a key transcription factor known to induce chondrogenesis, namely, Sox9, is highly expressed in our hADSCs/G-A-PAA bioconstruct, along with cartilage specific markers such as collagen type II, CEP68, and COMP extracellular matrix (ECM) components. These data suggest that our G-A-PAA structural properties and formulation might enable hADSCs conversion towards functional chondrocytes. We conclude that our novel G-A-PAA biomatrix is a good candidate for prospective in vivo CTE applications.

High Runx1 levels promote a reversible, more-differentiated cell state in hair-follicle stem cells during quiescence.

Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying matrix cells. EPs can revert to SCs upon injury, but whether this dedifferentiation occurs in normal HF homeostasis (hair cycle) and the mechanisms regulating both differentiation and dedifferentiation are unclear. Here, we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome toward a cell state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and dedifferentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche.