Single-Cell Profiling Reveals Global Immune Responses during the Progression of Murine Epidermal Neoplasms.

Immune cells determine the role of the tumor microenvironment during tumor progression, either suppressing tumor formation or promoting tumorigenesis. We analyzed the profile of immune cells in the tumor microenvironment of control mouse skins and skin tumors at the single-cell level. We identified 15 CD45 immune cell clusters, which broadly represent the most functionally characterized immune cell types including macrophages, Langerhans cells (LC), conventional type 1 dendritic cells (cDC1), conventional type 2 dendritic cells (cDC2), migratory/mature dendritic cells (mDC), dendritic epidermal T cells (DETC), dermal γδ T cells (γδT), T cells, regulatory T cells (Tregs), natural killer cells (NK), type 2 innate lymphoid cells (ILC2), neutrophils (Neu), mast cells (Mast), and two proliferating populations (Prolif.

Phosphodiesterase-4 Inhibitors Increase Pigment Cell Proliferation and Melanization in Cultured Melanocytes and within a 3-Dimensional Skin Equivalent Model.

Vitiligo is a common chronic autoimmune disease characterized by white macules and patches of the skin, having a negative impact on patients' life and without any definitive cure at present. Identification of new compounds to reverse depigmentation is therefore a pressing need for this disease. The pharmacologic compounds phosphodiesterase-4 inhibitors (PDE4is) are small molecules with immunomodulatory properties used for treatment of inflammatory dermatoses.

Vitiligo non-responding lesions to narrow band UVB have intriguing cellular and molecular abnormalities that may prevent epidermal repigmentation.

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions.

The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.

Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7).

Loricrin at the Boundary between Inside and Outside.

Cornification is a specialized mode of the cell-death program exclusively allowed for terrestrial amniotes. Recent investigations suggest that loricrin (LOR) is an important cornification effector. As the connotation of its name ("lorica" meaning an armor in Latin) suggests, the keratin-associated protein LOR promotes the maturation of the epidermal structure through organizing covalent cross-linkages, endowing the epidermis with the protection against oxidative injuries.

The Epidermis: Redox Governor of Health and Diseases.

A functional epithelial barrier necessitates protection against dehydration, and ichthyoses are caused by defects in maintaining the permeability barrier in the stratum corneum (SC), the uppermost protective layer composed of dead cells and secretory materials from the living layer stratum granulosum (SG). We have found that loricrin (LOR) is an essential effector of cornification that occurs in the uppermost layer of SG (SG1). LOR promotes the maturation of corneocytes and extracellular adhesion structure through organizing disulfide cross-linkages, albeit being dispensable for the SC permeability barrier.

Loricrin and NRF2 Coordinate Cornification.

Cornification involves cytoskeletal cross-linkages in corneocytes (the brick) and the secretion of lipids/adhesion structures to the interstitial space (the mortar). Because the assembly of lipid envelopes precedes corneocyte maturation, loricrin is supposed to be dispensable for the protection against desiccation. Although the phenotypes of knockout (LKO) mice are obscure, the antioxidative response on the KEAP1/NRF2 signaling pathway compensates for the structural defect in utero.

"Structural imprinting" of the cutaneous immune effector function.

Keratinization provides tolerance to desiccation and mechanical durability. Loricrin, which is an epidermal thiol-rich protein, efficiently stabilizes terminally differentiated keratinocytes and maintains redox homeostasis. The discovery of the largely asymptomatic loricrin knockout (LKO) phenotype decades ago was rather unpredicted.

Studying Immunotherapy Resistance in a Melanoma Autologous Humanized Mouse Xenograft.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME).

The KEAP1/NRF2 Signaling Pathway in Keratinization.

Keratinization is a tissue adaptation, but aberrant keratinization is associated with skin disorders such as ichthyoses, atopic dermatitis, psoriasis, and acne. The disease phenotype stems from the interaction between genes and the environment; therefore, an understanding of the adaptation machinery may lead to a new appreciation of pathomechanisms. The KEAP1/NRF2 signaling pathway mediates the environmental responses of squamous epithelial tissue.

Melanocyte Precursors in the Hair Follicle Bulge of Repigmented Vitiligo Skin Are Controlled by RHO-GTPase, KCTD10, and CTNNB1 Signaling.

In repigmentation of human vitiligo, the melanocyte (MC) precursors in the hair follicle bulge proliferate, migrate, and differentiate to repopulate the depigmented epidermis. Here, we present a comprehensive characterization of pathways and signals in the bulge that control the repigmentation process. Using biopsies from patients with vitiligo, we have selectively harvested, by laser capture microdissection, MC and keratinocyte precursors from the hair follicle bulge of untreated vitiligo skin and vitiligo skin treated with narrow-band UVB.

NRF2 Augments Epidermal Antioxidant Defenses and Promotes Atopy.

Atopic dermatitis is a chronic form of allergic contact dermatitis that is closely associated with a compromised epidermal barrier. Immunogenicity of a given electrophilic hapten after penetration of this barrier depends directly on biochemical reactions in the thiol-rich layer in the stratum granulosum. In response to electrophilic hapten, NF-erythroid 2-related factor 2 (NRF2) in keratinocytes efficiently induces the production of antioxidants.

Loricrin: Past, Present, and Future.

The terminal differentiation of the epidermis is a complex physiological process. During the past few decades, medical genetics has shown that defects in the stratum corneum (SC) permeability barrier cause a myriad of pathological conditions, ranging from common dry skin to lethal ichthyoses. Contrarily, molecular phylogenetics has revealed that amniotes have acquired a specialized form of cytoprotection cornification that provides mechanical resilience to the SC.

Tracing the Equilibrium Phase of Cancer Immunoediting in Epidermal Neoplasms via Longitudinal Intravital Imaging.

Recognition of transformed cells by the immune system can sometimes generate a rate-limiting equilibrium phase, wherein tumor outgrowth is prevented without complete neoplasm elimination. Targeting premalignancies during this immune-controlled bottleneck is a promising strategy for rational cancer prevention. Thus far, immune equilibrium has been difficult to model in a traceable way, and most immunoediting systems have been limited to mesenchymal tumor types.

Harnessing the Power of Regenerative Therapy for Vitiligo and Alopecia Areata.

Vitiligo and alopecia areata (AA) are common autoimmune conditions characterized by white spots on the skin (vitiligo) and bald spots on the scalp (AA), which significantly impact patients' lives by damaging their appearance and function. Melanocytes are the target of immune destruction in vitiligo and are hypothesized to be the site of immune attack in AA. This inflammatory process can be partially reversed by immunosuppressive drugs.

Mutations in Recessive Congenital Ichthyoses Illuminate the Origin and Functions of the Corneocyte Lipid Envelope.

The corneocyte lipid envelope (CLE), a monolayer of ω-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue.

High-efficiency RNA-based reprogramming of human primary fibroblasts.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts.

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the β-Catenin Pathway in the Hair Follicle Bulge Stem Cells.

Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin.