Intraoperative bioprinting of human adipose-derived stem cells and extra-cellular matrix induces hair follicle-like downgrowths and adipose tissue formation during full-thickness craniomaxillofacial skin reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2 % or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Apoptosis recognition receptors regulate skin tissue repair in mice.

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1+ macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for efferocytosis pathway genes and display altered efferocytosis signaling via the receptor Axl and its ligand Gas6. During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

Our skin is constantly exposed to potential damage from the outside world, and it is vital that any injuries are repaired quickly and effectively. Diabetes and many other health conditions can hamper wound healing, resulting in chronic wounds that are both painful and at risk of becoming infected, which can lead to serious illness and death of patients. After an injury to the skin, the wound becomes inflamed as immune cells rush to the site of injury to fight off infection and clear the wound of dead cells and debris. Some of these dead cells will have died by a highly controlled process known as apoptosis. These so-called apoptotic cells display signals on their surface that nearby healthy cells recognize. This triggers the healthy cells to eat the apoptotic cells to remove them from the wound. Previous studies have linked changes in cell death and the removal of dead cells to chronic wounds in patients with diabetes, but it remains unclear how removing dead cells from the wound affects healing. Justynski et al. used a genetic technique called single-cell RNA sequencing to study the patterns of gene activity in mouse skin cells shortly after a wound. The experiments found that, as the area around the wound started to become inflamed, the wounded cells produced signals of apoptosis that in turn triggered nearby healthy cells to remove them. Other signals relating to the removal of dead cells were also widespread in the mouse wounds and treating the wounds with drugs that inhibit these signals resulted in multiple defects in the healing process. Further experiments used the same approach to study samples of tissue taken from foot wounds in human patients with or without diabetes. This revealed that several genes involved in the removal of dead cells were more highly expressed in the wounds of diabetic patients than in the wounds of other individuals. These findings indicate that for wounds to heal properly it is crucial for the body to detect and clear apoptotic cells from the wound site. Further studies building on this work may help to explain why some diabetic patients suffer from chronic wounds and help to develop more effective treatments for them.

Intraoperative Bioprinting of Human Adipose-derived Stem cells and Extra-cellular Matrix Induces Hair Follicle-Like Downgrowths and Adipose Tissue Formation during Full-thickness Craniomaxillofacial Skin Reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2% or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo . Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Lineage commitment of dermal fibroblast progenitors is controlled by Kdm6b-mediated chromatin demethylation.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the epigenetic mechanisms that regulate DFP differentiation are not known. Our objective was to use multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanism that governs its differentiation potential. Our initial results indicated that the overall transcription profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage-specific genes. Surprisingly, the repressive chromatin profile of DFPs renders them unable to reform the skin in allograft assays despite their multipotent potential. We hypothesized that chromatin derepression was modulated by the H3K27me3 demethylase, Kdm6b/Jmjd3. Dermal fibroblast-specific deletion of Kdm6b/Jmjd3 in mice resulted in adipocyte compartment ablation and inhibition of mature dermal papilla functions, confirmed by additional single-cell RNA-seq, ChIP-seq, and allografting assays. We conclude that DFPs are functionally derepressed during murine skin development by Kdm6b/Jmjd3. Our studies therefore reveal a multimodal understanding of how DFPs differentiate into distinct fibroblast lineages and provide a novel publicly available multiomics search tool.

Lineage Commitment of Dermal Fibroblast Progenitors is Mediated by Chromatin De-repression.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the mechanisms that regulate lineage commitment of naive dermal progenitors to form niches around the hair follicle, dermis, and hypodermis, are unknown. In our study, we used multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanisms that govern its differentiation potential. Our results indicate that the overall chromatin profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage specific genes. Surprisingly, the repressed chromatin profile of DFPs renders them unable to reform skin in allograft assays despite their multipotent potential. Distinct fibroblast lineages, such as the dermal papilla and adipocytes contained specific chromatin profiles that were de-repressed during late embryogenesis by the H3K27-me3 demethylase, Kdm6b/Jmjd3. Tissue-specific deletion of Kdm6b/Jmjd3 resulted in ablating the adipocyte compartment and inhibiting mature dermal papilla functions in single-cell-RNA-seq, ChIPseq, and allografting assays. Altogether our studies reveal a mechanistic multimodal understanding of how DFPs differentiate into distinct fibroblast lineages, and we provide a novel multiomic search-tool within skinregeneration.org.

Parallel Single-Cell Multiomics Analysis of Neonatal Skin Reveals the Transitional Fibroblast States that Restrict Differentiation into Distinct Fates.

One of the keys to achieving skin regeneration lies within understanding the heterogeneity of neonatal fibroblasts, which support skin regeneration. However, the molecular underpinnings regulating the cellular states and fates of these cells are not fully understood. To investigate this, we performed a parallel multiomics analysis by processing neonatal murine skin for single-cell Assay for Transposase-Accessible Chromatin sequencing and single-cell RNA sequencing separately. Our approach revealed that fibroblast clusters could be sorted into papillary and reticular lineages on the basis of transcriptome profiling, as previously reported. However, single-cell Assay for Transposase-Accessible Chromatin sequencing analysis of neonatal fibroblast lineage markers, such as Dpp4/Cd26, Corin, and Dlk1 along with markers of myofibroblasts, revealed accessible chromatin in all fibroblast populations despite their lineage-specific transcriptome profiles. These results suggest that accessible chromatin does not always translate to gene expression and that many fibroblast lineage markers reflect a fibroblast state, which includes neonatal papillary fibroblasts, reticular fibroblasts, and myofibroblasts. This analysis also provides a possible explanation as to why these marker genes can be promiscuously expressed in different fibroblast populations under different conditions. Our single-cell Assay for Transposase-Accessible Chromatin sequencing analysis also revealed that the functional lineage restriction between dermal papilla and adipocyte fates is regulated by distinct chromatin landscapes. Finally, we have developed a webtool for our multiomics analysis: https://skinregeneration.org/scatacseq-and-scrnaseq-data-from-thompson-et-al-2021-2/.

Intra-Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction.

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.

Fibroblasts: Origins, definitions, and functions in health and disease.

Fibroblasts are diverse mesenchymal cells that participate in tissue homeostasis and disease by producing complex extracellular matrix and creating signaling niches through biophysical and biochemical cues. Transcriptionally and functionally heterogeneous across and within organs, fibroblasts encode regional positional information and maintain distinct cellular progeny. We summarize their development, lineages, functions, and contributions to fibrosis in four fibroblast-rich organs: skin, lung, skeletal muscle, and heart. We propose that fibroblasts are uniquely poised for tissue repair by easily reentering the cell cycle and exhibiting a reversible plasticity in phenotype and cell fate. These properties, when activated aberrantly, drive fibrotic disorders in humans.

The Three Rs of Single-Cell RNA Sequencing: Reuse, Refine, and Resource.

Single-cell RNA sequencing (scRNA-seq) provides an unprecedented ability to investigate cellular heterogeneity in entire organs and tissues, including human skin. Ascensión et al. (2020) combined and reanalyzed human skin scRNA-seq datasets to uncover new insights into fibroblast heterogeneity. This work demonstrates that new discoveries can be made from published data on the basis of principles of these three Rs: Reuse, Refine, and Resource.

Single-cell transcriptomic analysis of small and large wounds reveals the distinct spatial organization of regenerative fibroblasts.

Wound-induced hair follicle neogenesis (WIHN) has been an important model to study hair follicle regeneration during wound repair. However, the cellular and molecular components of the dermis that make large wounds more regenerative are not fully understood. Here, we compare and contrast recently published scRNA-seq data of small scarring wounds to wounds that regenerate in hope to elucidate the role of fibroblasts lineages in WIHN. Our analysis revealed an over-representation of the newly identified upper wound fibroblasts in regenerative wound conditions, which express the retinoic acid binding protein Crabp1. This regenerative cell type shares a similar gene signature to the murine papillary fibroblast lineage, which are necessary to support hair follicle morphogenesis and homeostasis. RNA velocity analysis comparing scarring and regenerating wounds revealed the divergent trajectories towards upper and lower wound fibroblasts and that the upper populations were closely associated with the specialized dermal papilla. We also provide analyses and explanation reconciling the inconsistency between the histological lineage tracing and the scRNA-seq data from recent reports investigating large wounds. Finally, we performed a computational test to map the spatial location of upper wound fibroblasts in large wounds which revealed that upper peripheral fibroblasts might harbour equivalent regenerative competence as those in the centre. Overall, our scRNA-seq reanalysis combining multiple samples suggests that upper wound fibroblasts are required for hair follicle regeneration and that papillary fibroblasts may migrate from the wound periphery to the centre during wound re-epithelialization. Moreover, data from this publication are made available on our searchable web resource: https://skinregeneration.org/.

Lef1 expression in fibroblasts maintains developmental potential in adult skin to regenerate wounds.

Scars are a serious health concern for burn victims and individuals with skin conditions associated with wound healing. Here, we identify regenerative factors in neonatal murine skin that transforms adult skin to regenerate instead of only repairing wounds with a scar, without perturbing development and homeostasis. Using scRNA-seq to probe unsorted cells from regenerating, scarring, homeostatic, and developing skin, we identified neonatal papillary fibroblasts that form a transient regenerative cell type that promotes healthy skin regeneration in young skin. These fibroblasts are defined by the expression of a canonical Wnt transcription factor Lef1 and using gain- and loss of function genetic mouse models, we demonstrate that Lef1 expression in fibroblasts primes the adult skin macroenvironment to enhance skin repair, including regeneration of hair follicles with arrector pili muscles in healed wounds. Finally, we share our genomic data in an interactive, searchable companion website (https://skinregeneration.org/). Together, these data and resources provide a platform to leverage the regenerative abilities of neonatal skin to develop clinically tractable solutions that promote the regeneration of adult tissue.

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called "horizontal whole mount," is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 µm-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

Dermal Blimp1 Acts Downstream of Epidermal TGFß and Wnt/ß-Catenin to Regulate Hair Follicle Formation and Growth.

B-lymphocyte-induced maturation protein 1 (Blimp1) is a transcriptional repressor that regulates cell growth and differentiation in multiple tissues, including skin. Although in the epidermis Blimp1 is important for keratinocyte and sebocyte differentiation, its role in dermal fibroblasts is unclear. Here we show that Blimp1 is dynamically regulated in dermal papilla cells during hair follicle (HF) morphogenesis and the postnatal hair cycle, preceding dermal Wnt/ß-catenin activation. Blimp1 ablation in E12.5 mouse dermal fibroblasts delayed HF morphogenesis and growth and prevented new HF formation after wounding. By combining targeted quantitative PCR screens with bioinformatic analysis and experimental validation we demonstrated that Blimp1 is both a target and a mediator of key dermal papilla inductive signaling pathways including transforming growth factor-ß and Wnt/ß-catenin. Epidermal overexpression of stabilized ß-catenin was able to override the HF defects in Blimp1 mutant mice, underlining the close reciprocal relationship between the dermal papilla and adjacent HF epithelial cells. Overall, our study reveals the functional role of Blimp1 in promoting the dermal papilla inductive signaling cascade that initiates HF growth.

The Sox2: GFP+/- knock-in mouse model does not faithfully recapitulate Sox2 expression in skin.

Hair follicle heterogeneity may be regulated by distinct dermal papillae (DP) that represent mesenchymal lineages, which can be defined by Sox2 expression. However, it was recently shown that GFP expression in the Sox2: GFP+/- mouse model occurs in the DPs of all hair follicle types, challenging the idea that hair follicle heterogeneity can be defined by DP heterogeneity. Here, we investigated whether the knock-in mouse model faithfully expresses GFP when compared to endogenous Sox2 expression. The results reveal that GFP expression is aberrant in both the infundibulum of hair follicles and in the DPs. Consequently, we provide an explanation for the aberrant expression of the knock-in gene based on the original cloning strategy for the mouse model in the context of a newly identified regulatory element associated within the coding region of Sox2.

Inhibition of ß-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing.

New hair follicles (HFs) do not form in adult mammalian skin unless epidermal Wnt signalling is activated genetically or within large wounds. To understand the postnatal loss of hair forming ability we monitored HF formation at small circular (2 mm) wound sites. At P2, new HFs formed in back skin, but HF formation was markedly decreased by P21. Neonatal tail also formed wound-associated HFs, albeit in smaller numbers. Postnatal loss of HF neogenesis did not correlate with wound closure rate but with a reduction in Lrig1-positive papillary fibroblasts in wounds. Comparative gene expression profiling of back and tail dermis at P1 and dorsal fibroblasts at P2 and P50 showed a correlation between loss of HF formation and decreased expression of genes associated with proliferation and Wnt/ß-catenin activity. Between P2 and P50, fibroblast density declined throughout the dermis and clones of fibroblasts became more dispersed. This correlated with a decline in fibroblasts expressing a TOPGFP reporter of Wnt activation. Surprisingly, between P2 and P50 there was no difference in fibroblast proliferation at the wound site but Wnt signalling was highly upregulated in healing dermis of P21 compared with P2 mice. Postnatal ß-catenin ablation in fibroblasts promoted HF regeneration in neonatal and adult mouse wounds, whereas ß-catenin activation reduced HF regeneration in neonatal wounds. Our data support a model whereby postnatal loss of hair forming ability in wounds reflects elevated dermal Wnt/ß-catenin activation in the wound bed, increasing the abundance of fibroblasts that are unable to induce HF formation.

ß-Catenin Stabilization in Skin Fibroblasts Causes Fibrotic Lesions by Preventing Adipocyte Differentiation of the Reticular Dermis.

The Wnt/ß-catenin pathway plays a central role in epidermal homeostasis and regeneration, but how it affects fibroblast fate decisions is unknown. We investigated the effect of targeted ß-catenin stabilization in dermal fibroblasts. Comparative gene expression profiling of stem cell antigen 1(-) (Sca1(-)) and Sca1(+) neonatal fibroblasts from upper and lower dermis, respectively, confirmed that Sca1(+) cells had a preadipocyte signature and showed differential expression of Wnt/ß-catenin-associated genes. By targeting all fibroblasts or selectively targeting Dlk1(+) lower dermal fibroblasts, we found that ß-catenin stabilization between developmental stages E16.5 and P2 resulted in a reduction in the dermal adipocyte layer with a corresponding increase in dermal fibrosis and an altered hair cycle. The fibrotic phenotype correlated with a reduction in the potential of Sca1(+) fibroblasts to undergo adipogenic differentiation ex vivo. Our findings indicate that Wnt/ß-catenin signaling controls adipogenic cell fate within the lower dermis, which potentially contributes to the pathogenesis of fibrotic skin diseases.

Compartmentalized Epidermal Activation of ß-Catenin Differentially Affects Lineage Reprogramming and Underlies Tumor Heterogeneity.

Wnt/ß-catenin activation in adult epidermis can induce new hair follicle formation and tumor development. We used lineage tracing to uncover the relative contribution of different stem cell populations. LGR6(+) and LRIG1(+) stem cells contributed to ectopic hair follicles formed in the sebaceous gland upon ß-catenin activation, whereas LGR5(+) cells did not. Lgr6, but not Lrig1 or Lgr5, was expressed in a subpopulation of interfollicular epidermal cells that were competent to form new hair follicles. Oncogenic ß-catenin expression in LGR5(+) cells led to formation of pilomatricomas, while LRIG1(+) cells formed trichoadenomas and LGR6(+) cells formed dermatofibromas. Tumor formation was always accompanied by a local increase in dermal fibroblast density and transient extracellular matrix remodeling. However, each tumor had a distinct stromal signature in terms of immune cell infiltrate and expression of CD26 and CD44. We conclude that compartmentalization of epidermal stem cells underlies different responses to ß-catenin and skin tumor heterogeneity.