The use of ectopic volar fibroblasts to modify skin identity.

Skin identity is controlled by intrinsic features of the epidermis and dermis and their interactions. Modifying skin identity has clinical potential, such as the conversion of residual limb and stump (nonvolar) skin of amputees to pressure-responsive palmoplantar (volar) skin to enhance prosthesis use and minimize skin breakdown. Greater keratin 9 (KRT9) expression, higher epidermal thickness, keratinocyte cytoplasmic size, collagen length, and elastin are markers of volar skin and likely contribute to volar skin resiliency. Given fibroblasts' capacity to modify keratinocyte differentiation, we hypothesized that volar fibroblasts influence these features. Bioprinted skin constructs confirmed the capacity of volar fibroblasts to induce volar keratinocyte features. A clinical trial of healthy volunteers demonstrated that injecting volar fibroblasts into nonvolar skin increased volar features that lasted up to 5 months, highlighting a potential cellular therapy.

Reassessing Long-Term Cryopreservation Strategies for Improved Quality, Safety, and Clinical Use of Allogeneic Dermal Progenitor Cells.

In regenerative medicine, ongoing advancements in cell culture techniques, including isolation, expansion, banking, and transport, are crucial for clinical success. Cryopreservation ensures off-the-freezer availability of living cells, enabling long-term storage and transport. Customizing cryopreservation techniques and cryoprotective agents (CPAs) for specific cell types is crucial for cell source quality, sustainability, safety, and therapeutic intervention efficiency. As regenerative medicine progresses, it becomes imperative that the scientific community and industry provide a comprehensive, cell-specific landscape of available and effective cryopreservation techniques, preventing trial-and-error approaches and unlocking the full potential of cell-based therapies. Open-sharing data could lead to safer, more efficient cell therapies and treatments. Two decades of dermal progenitor cell use for burn wound treatment and Good Manufacturing Practice-compliant technology transfers have highlighted the need for further cryopreservation optimization in manufacturing workflows. In this paper, we present experimental data assessing 5 different cryopreservation formulae for long-term storage of clinical-grade FE002 primary progenitor fibroblasts, emphasizing the crucial difference between DMSO-based and DMSO-free CPAs. Our findings suggest that CryoOx, a DMSO-free CPA, is a promising alternative yielding cell viability similar to that of established commercial CPAs. This research highlights the importance of secure, robust, and efficient cryopreservation techniques in cell banking for maximizing quality, ensuring patient safety, and advancing regenerative medicine.

Proteomic and secretomic comparison of young and aged dermal fibroblasts highlights cytoskeleton as a key component during aging.

Crucial for skin homeostasis, synthesis and degradation of extracellular matrix components are orchestrated by dermal fibroblasts. During aging, alterations of component expression, such as collagens and enzymes, lead to reduction of the mechanical cutaneous tension and defects of skin wound healing. The aim of this study was to better understand the molecular alterations underwent by fibroblasts during aging by comparing secretomic and proteomic signatures of fibroblasts from young (<35years) and aged (>55years) skin donors, in quiescence or TGF-stimulated conditions, using HLPC/MS. The comparison of the secretome from young and aged fibroblasts revealed that 16 proteins in resting condition, and 11 proteins after a 24h-lasting TGF-ß1-treatment, were expressed in significant different ways between the two cell groups (fold change>2, p-value <0.05), with a 77% decrease in the number of secreted proteins in aged cells. Proteome comparison between young and aged fibroblasts identified a significant change of 63 proteins in resting condition, and 73 proteins in TGF-ß1-stimulated condition, with a 67% increase in the number of proteins in aged fibroblasts. The majority of the differentially-expressed molecules belongs to the cytoskeleton-associated proteins and aging was characterized by an increase in Coronin 1C (CORO1C), and Filamin B (FLNB) expression in fibroblasts together with a decrease in Cofilin (CFL1), and Actin alpha cardiac muscle 1 (ACTC1) detection in aged cells, these proteins being involved in actin-filament polymerization and sharing co-activity in cell motility. Our present data reinforce knowledge about an age-related alteration in the synthesis of major proteins linked to the migratory and contractile functions of dermal human fibroblasts.

A Nonenzymatic Procedure to Obtain Human Mesenchymal Stromal Cells from the Dermis.

Human mesenchymal stromal cells (MSCs) have gained significant interest as cell-based therapeutics for organ restoration in the field of regenerative medicine. More recently, substantial attention has been directed toward cell-free therapy, achieved through the utilization of soluble factors possessing trophic and immunomodulatory properties present in the MSC secretome. This collection of soluble factors can be found either freely in the secretome or packed within its vesicular fraction, known as extracellular vesicles (EVs). MSCs can be derived from various tissue sources, each involving different extraction methods and yielding varying cell amounts. In this study, we describe a nonenzymatic procedure for a straightforward isolation of MSCs from the fetal dermis and the adult dermis. The results demonstrate the isolation of a cell population with a uniform MSC immunophenotype from the earliest passages (approximately 90% positive for the classical MSC markers CD90, CD105, and CD73, while negative for the hematopoietic markers CD34 and CD45, as well as HLA-DR). Additionally, we describe the procedures for cell expansion, banking, and secretome collection.

Effects of Nannochloropsis salina Fermented Oil on Proliferation of Human Dermal Papilla Cells and Hair Growth.

The hair follicle is the basis of hair regeneration, and the dermal papilla is one of the most important structures in hair regeneration. New intervention and reversal strategies for hair loss may arise due to the prevention of oxidative stress. GC/MS analysis was used to determine the compounds contained in NSO. Then, NSO was applied to DPC for cell proliferation and oxidative stress experiments. RNA-seq was performed in cells treated with NSO and minoxidil. The quantitative real-time polymerase chain reaction (qRT-PCR) was applied to verify the gene expression. The effects of NSO on hair length, weight, the number and depth of hair follicles, and the dermal thickness were also studied. GC/MS analysis showed that the main components of NSO were eicosapentaenoic acid, palmitic acid, and linoleic acid. NSO promotes DPC proliferation and reduces H2O2-mediated oxidative damage. NSO can also activate hair growth-related pathways and upregulate antioxidant-related genes analyzed by gene profiling. The topical application of NSO significantly promotes hair growth and increases hair length and weight in mice. NSO extract promotes hair growth and effectively inhibits oxidative stress, which is beneficial for the prevention and treatment of hair loss.

LincRNA-EPS Promotes Proliferation of Aged Dermal Fibroblast by Inducing CCND1.

The aging process is linked to numerous cellular changes, among which are modifications in the functionality of dermal fibroblasts. These fibroblasts play a crucial role in sustaining the healing of skin wounds. Reduced cell proliferation is a hallmark feature of aged dermal fibroblasts. Long intergenic non-coding RNA (lincRNAs), such as LincRNA-EPS (Erythroid ProSurvival), has been implicated in various cellular processes. However, its role in aged dermal fibroblasts and its impact on the cell cycle and its regulator, Cyclin D1 (CCND1), remains unclear. Primary dermal fibroblasts were isolated from the skin of 17-week-old (young) and 88-week-old (aged) mice. Overexpression of LincRNA-EPS was achieved through plasmid transfection. Cell proliferation was detected using the MTT assay. Real-time PCR was used to quantify relative gene expressions. Our findings indicate a noteworthy decline in the expression of LincRNA-EPS in aged dermal fibroblasts, accompanied by reduced levels of CCND1 and diminished cell proliferation in these aging cells. Significantly, the overexpression of LincRNA-EPS in aged dermal fibroblasts resulted in an upregulation of CCND1 expression and a substantial increase in cell proliferation. Mechanistically, LincRNA-EPS induces CCND1 expression by sequestering miR-34a, which was dysregulated in aged dermal fibroblasts, and directly targeting CCND1. These outcomes underscore the crucial role of LincRNA-EPS in regulating CCND1 and promoting cell proliferation in aged dermal fibroblasts. Our study provides novel insights into the molecular mechanisms underlying age-related changes in dermal fibroblasts and their implications for skin wound healing. The significant reduction in LincRNA-EPS expression in aged dermal fibroblasts and its ability to induce CCND1 expression and enhance cell proliferation highlight its potential as a therapeutic target for addressing age-related skin wound healing.

Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

Design and evaluation of a bilayered dermal/hypodermal 3D model using a biomimetic hydrogel formulation.

Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Hair Growth Promoting Effects of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor in Human Follicle Dermal Papilla Cells.

Prostaglandin E2 (PGE2) is known to be effective in regenerating tissues, and bimatoprost, an analog of PGF2α, has been approved by the FDA as an eyelash growth promoter and has been proven effective in human hair follicles. Thus, to enhance PGE2 levels while improving hair loss, we found dihydroisoquinolinone piperidinylcarboxy pyrazolopyridine (DPP), an inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using DeepZema®, an AI-based drug development program. Here, we investigated whether DPP improved hair loss in human follicle dermal papilla cells (HFDPCs) damaged by dihydrotestosterone (DHT), which causes hair loss. We found that DPP enhanced wound healing and the expression level of alkaline phosphatase in DHT-damaged HFDPCs. We observed that DPP significantly down-regulated the generation of reactive oxygen species caused by DHT. DPP recovered the mitochondrial membrane potential in DHT-damaged HFDPCs. We demonstrated that DPP significantly increased the phosphorylation levels of the AKT/ERK and activated Wnt signaling pathways in DHT-damaged HFDPCs. We also revealed that DPP significantly enhanced the size of the three-dimensional spheroid in DHT-damaged HFDPCs and increased hair growth in ex vivo human hair follicle organ culture. These data suggest that DPP exhibits beneficial effects on DHT-damaged HFDPCs and can be utilized as a promising agent for improving hair loss.

Mmp14-dependent remodeling of the pericellular-dermal collagen interface governs fibroblast survival.

Dermal fibroblasts deposit type I collagen, the dominant extracellular matrix molecule found in skin, during early postnatal development. Coincident with this biosynthetic program, fibroblasts proteolytically remodel pericellular collagen fibrils by mobilizing the membrane-anchored matrix metalloproteinase, Mmp14. Unexpectedly, dermal fibroblasts in Mmp14-/- mice commit to a large-scale apoptotic program that leaves skin tissues replete with dying cells. A requirement for Mmp14 in dermal fibroblast survival is recapitulated in vitro when cells are embedded within, but not cultured atop, three-dimensional hydrogels of crosslinked type I collagen. In the absence of Mmp14-dependent pericellular proteolysis, dermal fibroblasts fail to trigger ß1 integrin activation and instead actuate a TGF-ß1/phospho-JNK stress response that leads to apoptotic cell death in vitro as well as in vivo. Taken together, these studies identify Mmp14 as a requisite cell survival factor that maintains dermal fibroblast viability in postnatal dermal tissues.

MicroRNA-181a Targets GNAI2 and Affects the Proliferation and Induction Ability of Dermal Papilla Cells: The Potential Involvement of the Wnt/ß-Catenin Signaling Pathway.

Wool is generated by hair follicles (HFs), which are crucial in defining the length, diameter, and morphology of wool fibers. However, the regulatory mechanism of HF growth and development remains largely unknown. Dermal papilla cells (DPCs) are a specialized cell type within HFs that play a crucial role in governing the growth and development of HFs. This study aims to investigate the proliferation and induction ability of ovine DPCs to enhance our understanding of the potential regulatory mechanisms underlying ovine HF growth and development. Previous research has demonstrated that microRNA-181a (miR-181a) was differentially expressed in skin tissues with different wool phenotypes, which indicated that miR-181a might play a crucial role in wool morphogenesis. In this study, we revealed that miR-181a inhibited the proliferation and induction ability of ovine DPCs by quantitative Real-time PCR (qRT-PCR), cell counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and alkaline phosphatase staining. Then, we also confirmed G protein subunit alpha i2 (GNAI2) is a target gene of miR-181a by dual luciferase reporter assay, qRT-PCR, and Western blot, and that it could promote the proliferation and induction ability of ovine DPCs. In addition, GNAI2 could also activate the Wnt/ß-Catenin signaling pathway in ovine DPCs. This study showed that miR-181a can inhibit the proliferation and induction ability of ovine DPCs by targeting GNAI2 through the Wnt/ß-Catenin signaling pathway.

Age and sex effects on histological features and in vitro culturing of Antillean manatee (Trichechus manatus manatus, Linnaeus 1758) dermis.

The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and in vitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.

Combination of Autophagy and Stem Cell Enhancing Properties of Natural Product Extracts in Human Dermal Papilla Stem Cells.

BACKGROUND/AIM: Dermal papilla (DP) stem cells are known for their remarkable regenerative capacity, making them a valuable model for assessing the effects of natural products on cellular processes, including stemness, and autophagy. MATERIALS AND METHODS: Autophagy and stemness characteristics were assessed using real-time RT-PCR to analyze mRNA levels, along with immunofluorescence and western blot techniques for protein level evaluation. RESULTS: Butterfly Pea, Emblica Fruits, Kaffir Lime, and Thunbergia Laurifolia extracts induced autophagy in DP cells. Kaffir Lime-treated cells exhibited increase in the OCT4, NANOG, and SOX2 mRNA (6-, 5, and 5.5-fold, respectively), and protein levels (4-, 3-, and 1.5-fold, respectively). All extracts activated the survival protein kinase B (Akt) in DP cells. CONCLUSION: Natural products are a promising source for promoting hair growth by rejuvenating hair stem cells.

Characteristics of tunneling nanotube-like structures formed by human dermal microvascular pericytes in vitro.

Tunneling nanotubes (TNTs) represent an innovative way for cells to communicate with one another, as they act as long conduits between cells. However, their roles in human dermal microvascular pericytes (HDMPCs) interaction remain elusive in vitro. In this work, we identified and characterized the TNT-like structures that connected two or more pericytes in two-dimensional cultures and formed a functional network in the human dermis. Immunofluorescence assay indicated that the F-actin was an essential element to form inter-pericyte TNT-like structures, as it decreased in actin polymer inhibitor-cytochalasin B treated groups, and microtubules were present in almost half of the TNT-like structures. Most importantly, we only found the presence of mitochondrial in TNT-like structures containing α-tubulin, and the application of microtubule assembly inhibitor-Nocodazole significantly reduced the percentage of TNT-like structures that contain α-tubulin, resulting in a sudden decrease in the positive rate of cytochrome c oxidase subunit 4 isoform 1 (COX IV, a marker of mitochondria) in TNT-like structures. In summary, we described a novel intercellular communication-TNT-like structures-between HDMPCs in vitro, and this work allows us to properly understand the cellular mechanisms of spreading materials between HDMPCs, shedding light on the role of HDMPCs.

Immunohistochemical-properties of the dermal embryonic telocytes.

The current investigation aims to study the embryonic dermis formed in the early stages of development and identify the initial interstitial components of the dermis that serve as biological and structural scaffolds for the development of the dermal tissue. To investigate the dermal structure, the current study used morphological and immunological techniques. TCs identified by TEM. They had a cell body and unique podomeres and podoms. They formed a 3D network spread throughout the dermis. Homocellular contact established between them, as well as heterocellular contacts with other cells. Immunohistochemical techniques using specific markers for TCss CD34, CD117, and VEGF confirmed TC identification. TCs represent the major interstitial component in the dermal tissue. They established a 3D network, enclosing other cells and structures. Expression of VEGF by TC promotes angiogenesis. TCs establish cellular contact with sprouting endothelial cells. At the site of cell junction with TCs, cytoskeletal filaments identified and observed to form the pseudopodium core that projects from endothelial cells. TCs had proteolytic properties that expressed MMP-9, CD68, and CD21. Proteolytic activity aids in the removal of components of the extracellular matrix and the phagocytosis of degraded remnants to create spaces to facilitate the development of new dermal structures. In conclusion, TCs organized the scaffold for the development of future dermal structures, including fibrous components and skin appendages. Studying dermal TCs would be interested in the possibility of developing therapeutic strategies for treating different skin disorders and diseases.

Human dermal fibroblast-derived secretory proteins for regulating nerve restoration: A bioinformatic approach.

BACKGROUND: Human dermal fibroblasts secrete diverse proteins that regulate wound repair and tissue regeneration. METHODS: In this study, dermal fibroblast-conditioned medium (DFCM) proteins potentially regulating nerve restoration were bioinformatically selected among the 337 protein lists identified by quantitative liquid chromatography-tandem mass spectrometry. Using these proteins, protein-protein interaction network analysis was conducted. In addition, the roles of DFCM proteins were reviewed according to their protein classifications. RESULTS: Gene Ontology protein classification categorized these 57 DFCM proteins into various classes, including protein-binding activity modulator (N = 11), cytoskeletal protein (N = 8), extracellular matrix protein (N = 6), metabolite interconversion enzyme (N = 5), chaperone (N = 4), scaffold/adapter protein (N = 4), calcium-binding protein (N = 3), cell adhesion molecule (N = 2), intercellular signal molecule (N = 2), protein modifying enzyme (N = 2), transfer/carrier protein (N = 2), membrane traffic protein (N = 1), translational protein (N = 1), and unclassified proteins (N = 6). Further protein-protein interaction network analysis of 57 proteins revealed significant interactions among the proteins that varied according to the settings of confidence score. CONCLUSIONS: Our bioinformatic analysis demonstrated that DFCM contains many secretory proteins that form significant protein-protein interaction networks crucial for regulating nerve restoration. These findings underscore DFCM proteins' critical roles in various nerve restoration stages during the wound repair process.

Hair growth-promoting effects of Enz_MoriL on human dermal papilla cells through modulation of the Wnt/ß-Catenin and JAK-STAT signaling pathways.

Enz_MoriL is a naturally occurring substance extracted from the leaves of Morus alba L. through enzymatic conversion. Historically, M. alba L. has been recognized for its potential to promote hair regrowth. However, the precise mechanism by which Enz_MoriL affects human hair follicle dermal papilla cells (hDPCs) remains unclear. The aim of this study was to investigate the molecular basis of Enz_MoriL's effect on hair growth in hDPCs. Interferon-gamma (IFN-γ) was used to examine the effects of Enz_MoriL on hDPCs during the anagen and catagen phases, as well as under conditions mimicking alopecia areata (AA). Enz_MoriL demonstrated the ability to promote cell proliferation in both anagen and catagen stages. It increased the levels of active ß-catenin in the catagen stage induced by IFN-γ, leading to its nuclear translocation. This effect was achieved by increasing the phosphorylation of GSK3ß and decreasing the expression of DKK-1. This stimulation induced proliferation in hDPCs and upregulated the expression of the Wnt family members 3a, 5a, and 7a at the transcript level. Additionally, Enz_MoriL suppressed JAK1 and STAT3 phosphorylation, contrasting with IFN-γ, which induced them in the catagen stage. In conclusion, Enz_MoriL directly induced signals for anagen re-entry into hDPCs by affecting the Wnt/ß-catenin pathway and enhancing the production of growth factors. Furthermore, Enz_MoriL attenuated and reversed the interferon-induced AA-like environment by blocking the JAK-STAT pathway in hDPCs.

Dental Pulp Stem Cells Modulate Inflammasome Pathway and Collagen Deposition of Dermal Fibroblasts.

Fibrosis is a pathological condition consisting of a delayed deposition and remodeling of the extracellular matrix (ECM) by fibroblasts. This deregulation is mostly triggered by a chronic stimulus mediated by pro-inflammatory cytokines, such as TNF-α and IL-1, which activate fibroblasts. Due to their anti-inflammatory and immunosuppressive potential, dental pulp stem cells (DPSCs) could affect fibrotic processes. This study aims to clarify if DPSCs can affect fibroblast activation and modulate collagen deposition. We set up a transwell co-culture system, where DPSCs were seeded above the monolayer of fibroblasts and stimulated with LPS or a combination of TNF-α and IL-1ß and quantified a set of genes involved in inflammasome activation or ECM deposition. Cytokines-stimulated co-cultured fibroblasts, compared to unstimulated ones, showed a significant increase in the expression of IL-1ß, IL-6, NAIP, AIM2, CASP1, FN1, and TGF-ß genes. At the protein level, IL-1ß and IL-6 release as well as FN1 were increased in stimulated, co-cultured fibroblasts. Moreover, we found a significant increase of MMP-9 production, suggesting a role of DPSCs in ECM remodeling. Our data seem to suggest a crosstalk between cultured fibroblasts and DPSCs, which seems to modulate genes involved in inflammasome activation, ECM deposition, wound healing, and fibrosis.

YAP prevents senescence of dermal fibroblast and inhibits melanogenesis via paracrine effect of DKK1.

Senile skin hyperpigmentation displays remarkable histopathological features of dermal aging. The crosstalk between melanocytes and dermal fibroblasts plays crucial roles in aging-related pigmentation. While senescent fibroblasts can upregulate pro-melanogenic factors, the role of anti-melanogenic factors, such as dickkopf1 (DKK1), and the upstream regulatory mechanism during aging remain obscure. This study investigated the roles of yes-associated protein (YAP) and DKK1 in the regulation of dermal fibroblast senescence and melanogenesis. Our findings demonstrated decreased YAP activity and DKK1 levels in intrinsic and extrinsic senescent fibroblasts. YAP depletion induced fibroblast senescence and downregulated the expression and secretion of DKK1, whereas YAP overexpression partially reversed the effect. The transcriptional regulation of DKK1 by YAP was supported by dual-luciferase reporter and chromatin immunoprecipitation assays. Moreover, YAP depletion in fibroblasts upregulated Wnt/ß-catenin in melanocytes and stimulated melanogenesis, which was partially rescued by the re-supplementation of DKK1. Conversely, overexpression of YAP in senescent fibroblasts decreased Wnt/ß-catenin levels in melanocytes and inhibited melanogenesis. Additionally, reduced levels of YAP and DKK1 were verified in the dermis of solar lentigines. These findings suggest that, during skin aging, epidermal pigmentation may be influenced by YAP in the dermal microenvironment via the paracrine effect of DKK1.