3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

Melanocyte stem cells in the skin: Origin, biological characteristics, homeostatic maintenance and therapeutic potential.

Melanocyte stem cells (MSCs), melanocyte lineage-specific skin stem cells derived from the neural crest, are observed in the mammalian hair follicle, the epidermis or the sweat gland. MSCs differentiate into mature melanin-producing melanocytes, which confer skin and hair pigmentation and uphold vital skin functions. In controlling and coordinating the homeostasis, repair and regeneration of skin tissue, MSCs play a vital role. Decreased numbers or impaired functions of MSCs are closely associated with the development and therapy of many skin conditions, such as hair graying, vitiligo, wound healing and melanoma. With the advancement of stem cell technology, the relevant features of MSCs have been further elaborated. In this review, we provide an exhaustive overview of cutaneous MSCs and highlight the latest advances in MSC research. A better understanding of the biological characteristics and micro-environmental regulatory mechanisms of MSCs will help to improve clinical applications in regenerative medicine, skin pigmentation disorders and cancer therapy. KEY POINTS: This review provides a concise summary of the origin, biological characteristics, homeostatic maintenance and therapeutic potential of cutaneous MSCs. The role and potential application value of MSCs in skin pigmentation disorders are discussed. The significance of single-cell RNA sequencing, CRISPR-Cas9 technology and practical models in MSCs research is highlighted.

The HOCl dry fog-is it safe for human cells?

This study aims to investigate if high-concentration HOCl fogging disinfection causes cytotoxicity and genotoxicity to cultured primary human skin fibroblasts. The cells were exposed to a dry fog of HOCl produced from solutions with a concentration of 300 ppm (5.72 mM) or 500 ppm (9.53 mM). After four times when fibroblasts were exposed to aerosolized HOCl at a concentration of 500 ppm for 9 minutes, significant cytotoxicity and genotoxicity effects were observed. Significant changes in the morphology of fibroblasts and cell death due to membrane disruption were observed, independent of the number of exposures. Flow cytometry analyses performed under these experimental conditions indicated a decrease in the number of cells with an intact cell membrane in the exposed samples compared to the sham samples, dropping to 49.1% of the total cells. Additionally, under the same conditions, the neutral comet assay results demonstrated significant DNA damage in the exposed cells. However, no analogous damages were found when the cells were exposed to aerosolized HOCl generated from a 300-ppm solution for 3 minutes, whether once or four times. Therefore, we have concluded that aerosolized HOCl in dry fog, with a concentration exceeding 300 ppm, can cause cytotoxic and genotoxic effects on human skin fibroblasts.

Genetic Tools for Cell Lineage Tracing and Profiling Developmental Trajectories in the Skin.

The epidermis is the body's first line of protection against dehydration and pathogens, continually regenerating the outermost protective skin layers throughout life. During both embryonic development and wound healing, epidermal stem and progenitor cells must respond to external stimuli and insults to build, maintain, and repair the cutaneous barrier. Recent advances in CRISPR-based methods for cell lineage tracing have remarkably expanded the potential for experiments that track stem and progenitor cell proliferation and differentiation over the course of tissue and even organismal development. Additional tools for DNA-based recording of cellular signaling cues promise to deepen our understanding of the mechanisms driving normal skin morphogenesis and response to stressors as well as the dysregulation of cell proliferation and differentiation in skin diseases and cancer. In this review, we highlight cutting-edge methods for cell lineage tracing, including in organoids and model organisms, and explore how cutaneous biology researchers might leverage these techniques to elucidate the developmental programs that support the regenerative capacity and plasticity of the skin.

Skin graft with dermis and appendages generated in vivo by cell competition.

Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

Cell Senescence-Independent Changes of Human Skin Fibroblasts with Age.

Skin ageing is defined, in part, by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that in vitro senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with previous reports of senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that the increased collagen degradation of aged fibroblasts might reflect senescence, the reduced collagen production likely reflects senescence-independent processes.

Rh family C glycoprotein contributes to psoriatic inflammation through regulating the dysdifferentiation and cytokine secretion of keratinocytes.

BACKGROUND: Keratinocyte dysdifferentiation and proinflammatory cytokine production play a central role in psoriatic inflammation. According to recent studies, the Rh family C glycoprotein (RHCG) enhances cell proliferation and disrupts cell differentiation. However, the specific role of RHCG psoriasis development remains unclear. OBJECTIVE: We here explored the effect of RHCG on keratinocytes under psoriatic inflammation. METHODS: The cell counting kit­8 assay was conducted to assess proliferation. RHCG protein expression was assessed through western blotting and enzyme-linked immunosorbent assays. The expression of proinflammatory cytokines and differentiation markers was analyzed through a quantitative reverse-transcription polymerase chain reaction. RESULTS: Both RHCG mRNA and protein levels increased in psoriatic skin. Notably, cultured keratinocytes treated with an M5 cocktail, which mimics psoriatic inflammation, exhibited higher RHCG expression. Furthermore, RHCG overexpression promoted keratinocyte proliferation, accompanied by an increase in the production of interleukin (IL)-1ß, IL-6, and IL-8, and tumor necrosis factor-α. RHCG overexpression also resulted in higher expression of keratin 17, a differentiation marker. Conversely, RHCG gene knockdown reduced keratinocyte proliferation and cytokine secretion. RHCG inhibition in cells recovered both keratin 1 and loricrin expression. Additionally, RHCG overexpression facilitated the phosphorylation of nuclear factor-kappa B and extracellular signal-regulated protein kinase signaling pathways. Importantly, when these signaling pathways were inhibited, the effect of RHCG on keratinocytes was attenuated. CONCLUSION: These findings support the substantial role of RHCG in psoriatic inflammation development and suggest that RHCG serves as a potential target for psoriasis treatment.

Generation and characterization of an iPS cell line (PUMCi006-A) from skin fibroblasts of a patient with an M239T mutation in PSEN2 gene.

Presenilin-2 (PSEN2) mutation is one of the pathogenic factors of autosomal dominant early-onset Alzheimer's disease (EOAD). We generated a human induced pluripotent stem cell (iPSC) line from fibroblasts of an EOAD patient carrying PSEN2 mutation (c.716 T > C) utilizing Sendai reprogramming kit. The resulting iPSC line carried patient-specific point mutation, exhibited typical iPSC morphology, retained a normal karyotype, expressed pluripotency markers, and could form embryoid bodies. Established iPSC line serve as valuable resource for EOAD disease pathogenesis modelling and drug screening.

KTN1 mediated unfolded protein response protects keratinocytes from ionizing radiation-induced DNA damage.

BACKGROUND: The unfolded protein response (UPR) is one of the cytoprotective mechanisms against various stresses and essential for the normal function of skin. Skin injury caused by ionizing radiation (IR) is a common side effect of radiotherapy and it is unclear how UPR affects IR-induced skin injury. OBJECTIVES: To verify the effect of UPR on IR-induced DNA damage in keratinocytes and the relation between an endoplasmic reticulum (ER) protein KTN1 and UPR. METHODS: All experiments were performed on keratinocytes models: HaCaT and HEK-A. ER lumen and the expression levels of KTN1 and UPR pathway proteins (PERK, IRE1α and ATF6) were examined by transmission electron microscopy and immunoblotting, respectively. 4-PBA, an UPR inhibitor, was used to detected its effects on DNA damage and cell proliferation. Subsequently, the effects of KTN1 deletion on UPR, DNA damage and cell proliferation after IR were detected. Tunicamycin was used to reactivate UPR and then we examined its effects on DNA damage. RESULTS: UPR was activated by IR in keratinocytes. Inhibition of UPR aggravated DNA damage and suppressed cell proliferation after IR. KTN1 expression was upregulated by IR and KTN1 depletion reduced ER expansion and the expression of UPR-related proteins. Moreover, KTN1 depletion aggravated DNA damage and suppressed cell proliferation after IR could reversed by reactivation of UPR. CONCLUSION: KTN1 deletion aggravates IR-induced keratinocyte DNA damage via inhibiting UPR. Our findings provide new insights into the mechanisms of keratinocytes in response to IR-induced damage.

Rosemary extract and rosmarinic acid accelerate elastic fiber formation by increasing the expression of elastic fiber components in dermal fibroblasts.

Ultraviolet (UV)-induced skin photoaging is caused by qualitative and quantitative degradation of dermal extracellular matrix components such as collagen and elastic fibers. Elastic fibers are important for maintaining cutaneous elasticity, despite their small amount in the skin. Previously, microfibril-associated protein 4 (MFAP-4), which is downregulated in photoaging dermis, has been found to be essential for elastic fiber formation by interaction with both fibrillin-1 and elastin, which are core components of elastic fiber. In addition, enhanced cutaneous MFAP-4 expression in a human skin-xenografted murine photoaging model protects against UV-induced photodamage accompanied by the prevention of elastic fiber degradation and aggravated elasticity. We therefore hypothesized that the upregulation of MFAP-4 in dermal fibroblasts may more efficiently accelerate elastic fiber formation. We screened botanical extracts for MFAP-4 expression-promoting activity in normal human dermal fibroblasts (NHDFs). We found that rosemary extract markedly promotes early microfibril formation and mature elastic fiber formation along with a significant upregulation of not only MFAP-4 but also fibrillin-1 and elastin in NHDFs. Furthermore, rosmarinic acid, which is abundant in rosemary extract, accelerated elastic fiber formation via upregulation of transforming growth factor ß-1. This was achieved by the induction of cAMP response element-binding protein phosphorylation, demonstrating that rosmarinic acid represents one of the active ingredients in rosemary extract. Based on the findings in this study, we conclude that rosemary extract and rosmarinic acid represent promising materials that exert a preventive or ameliorative effect on skin photoaging by accelerating elastic fiber formation.

Water-soluble intracellular extract of Desmodesmus sp. YT enhanced the antioxidant capacity of human skin fibroblast to protect the skin from UV damage.

BACKGROUND: The oxidative stress induced by ultraviolet (UV) radiation is a pivotal factor in skin aging and can even contribute to the development of skin cancer. AIM: This study explored the antioxidant effect and mechanism of water-soluble intracellular extract (WIE) of Desmodesmus sp.YT (YT), aiming to develop a natural antioxidant suitable for incorporation into cosmetics. METHODS: The study evaluated the scavenging capacity of YT-WIE against free radicals and assessed its impact on human skin fibroblasts (HSF) cell viability and UV resistance using Cell Counting Kit-8 (CCK-8). Transcriptome sequencing was employed to elucidate the mechanism of action, while RT-qPCR and western blot were used to validate the expression of key genes. RESULTS: YT-WIE displayed robust antioxidant activity, demonstrating potent scavenging abilities against 2,2-diphenyl-1-picrylhydrazyl (DPPH; IC50 = 0.55 mg mL-1), 2,2'-Azino-bis (3 ethylbenzothiazoline-6-sulfonic acid; ABTS; IC50 = 3.11 mg mL-1), Hydroxyl (·OH; IC50 = 2.21 mg mL-1), and Superoxide anion (O2 •-; IC50 = 0.98 mg mL-1). Furthermore, compared to the control group, the YT-WIE group exhibited an 89.30% enhancement in HSF viability and a 44.63% increase in survival rate post-UV irradiation. Significant upregulation of antioxidant genes (GCLC, GCLM, TXNRD1, HMOX1, NQO1) was observed with YT-WIE treatment at 400 µg mL-1, with fold increases ranging from 1.13 to 5.85 times. CONCLUSION: YT-WIE demonstrated considerable potential as an antioxidant, shielding human cells from undue oxidative stress triggered by external stimuli such as UV radiation. This suggests its promising application in cosmetics antioxidants.

Effects of pulsed electrical stimulation on α-smooth muscle actin and type I collagen expression in human dermal fibroblasts.

Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.

Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

Automated Whole Slide Imaging for Label-Free Histology Using Photon Absorption Remote Sensing Microscopy.

OBJECTIVE: Pathologists rely on histochemical stains to impart contrast in thin translucent tissue samples, revealing tissue features necessary for identifying pathological conditions. However, the chemical labeling process is destructive and often irreversible or challenging to undo, imposing practical limits on the number of stains that can be applied to the same tissue section. Here we present an automated label-free whole slide scanner using a PARS microscope designed for imaging thin, transmissible samples. METHODS: Peak SNR and in-focus acquisitions are achieved across entire tissue sections using the scattering signal from the PARS detection beam to measure the optimal focal plane. Whole slide images (WSI) are seamlessly stitched together using a custom contrast leveling algorithm. Identical tissue sections are subsequently H&E stained and brightfield imaged. The one-to-one WSIs from both modalities are visually and quantitatively compared. RESULTS: PARS WSIs are presented at standard 40x magnification in malignant human breast and skin samples. We show correspondence of subcellular diagnostic details in both PARS and H&E WSIs and demonstrate virtual H&E staining of an entire PARS WSI. The one-to-one WSI from both modalities show quantitative similarity in nuclear features and structural information. CONCLUSION: PARS WSIs are compatible with existing digital pathology tools, and samples remain suitable for histochemical, immunohistochemical, and other staining techniques. SIGNIFICANCE: This work is a critical advance for integrating label-free optical methods into standard histopathology workflows.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Angiogenin as a Possible Mediator of Macrophage-Mediated Regulation of Fibroblast Functions.

We studied angiogenin production by human macrophages and evaluated the role of this factor in the macrophage-mediated regulation of fibroblasts. All macrophage subtypes, and especially the efferocytosis-polarized macrophages, M2(LS), actively produced angiogenin. Exogenous recombinant angiogenin dose-dependently enhanced the proliferation and differentiation of dermal fibroblasts. The addition of the angiogenin inhibitor to fibroblasts cultures suppressed the stimulating effect of exogenous angiogenin or M2(LS) conditioned media. These findings indicate the involvement of angiogenin in the macrophage-mediated paracrine regulation of skin fibroblasts.

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several B cell malignancies and Kaposi's sarcoma. We analyzed the function of K8.1, the major antigenic component of the KSHV virion in the infection of different cells. To do this, we deleted K8.1 from the viral genome. It was found that K8.1 is critical for the infection of certain epithelial cells, e.g., a skin model cell line but not for infection of many other cells. K8.1 was found to mediate attachment of the virus to cells where it plays a role in infection. In contrast, we did not find K8.1 or a related protein from a closely related monkey virus to activate fusion of the viral and cellular membranes, at least not under the conditions tested. These findings suggest that K8.1 functions in a highly cell-specific manner during KSHV entry, playing a crucial role in the attachment of KSHV to, e.g., skin epithelial cells.

Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Calling long distance: Cell cycle-dependent Ca2+ flows connect stem cells across regeneration tissues.

How adult stem cells signal in vivo over time to coordinate their fate and behavior across self-renewing tissues remains a challenging question. In this issue, Moore et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202302095) combine high-resolution live imaging in mice with machine learning tools to reveal temporally regulated tissue-scale patterns of Ca2+ signaling orchestrated by cycling basal stem cells of the skin epidermis.