Hair Germ Model In Vitro via Human Postnatal Keratinocyte-Dermal Papilla Interactions: Impact of Hyaluronic Acid

Hair follicle (HF) reconstruction in vitro is a promising field in alopecia treatment and human HF development research. Here, we combined postnatal human dermal papilla (DP) cells and skin epidermal keratinocytes (KCs) in a hanging drop culture to develop an artificial HF germ. The method is based on DP cell hair-inducing properties and KC self-organization. We evaluated two protocols of aggregate assembling. Mixed HF germ-like structures demonstrated the initiation of epithelial-mesenchymal interaction, including WNT pathway activation and expression of follicular markers. We analyzed the influence of possible DP cell niche components including soluble factors and extracellular matrix (ECM) molecules in the process of the organoid assembling and growth. Our results demonstrated that soluble factors had little impact on HF germ generation and Ki67+ cell score inside the organoids although BMP6 and VD3 maintained effectively the DP identity in the monolayer culture. Aggrecan, biglycan, fibronectin, and hyaluronic acid (HA) significantly stimulated cell proliferation in DP cell monolayer culture without any effect on DP cell identity. Most of ECM compounds prevented the formation of cell aggregates while HA promoted the formation of larger organoids. In conclusion, our model could be suitable to study cell-cell and cell-niche interactions during HF reconstruction in vitro

Extracellular Matrix as a Regulator of Epidermal Stem Cell Fate

Epidermal stem cells reside within the specific anatomic location, called niche, which is a microenvironment that interacts with stem cells to regulate their fate. Regulation of many important processes, including maintenance of stem cell quiescence, self-renewal, and homeostasis, as well as the regulation of division and differentiation, are common functions of the stem cell niche. As it was shown in multiple studies, extracellular matrix (ECM) contributes a lot to stem cell niches in various tissues, including that of skin. In epidermis, ECM is represented, primarily, by a highly specialized ECM structure, basement membrane (BM), which separates the epidermal and dermal compartments. Epidermal stem cells contact with BM, but when they lose the contact and migrate to the overlying layers, they undergo terminal differentiation. When considering all of these factors, ECM is of fundamental importance in regulating epidermal stem cells maintenance, proper mobilization, and differentiation. Here, we summarize the remarkable progress that has recently been made in the research of ECM role in regulating epidermal stem cell fate, paying special attention to the hair follicle stem cell niche. We show that the destruction of ECM components impairs epidermal stem cell morphogenesis and homeostasis. A deep understanding of ECM molecular structure as well as the development of in vitro system for stem cell maintaining by ECM proteins may bring us to developing new approaches for regenerative medicine

Expression of the Antimicrobial Peptide SE-33-A2P, a Modified Analog of Cathelicidin, and an Analysis of Its Properties

In this study, we developed a method for the expression of the antimicrobial peptide SE-33-A2P in E. coli bacterial cells. The SE-33-A2P peptide consists of A2P and SE-33 peptides and is a retro analog of cathelicidin possessing antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, the A2P peptide is a self-cleaving peptide. For an efficient expression of the SE-33-A2P peptide, a gene encoding several repetitive sequences of the SE-33 peptide separated by A2P sequences was created. The gene was cloned into a plasmid, with which E. coli cells were transformed. An induction of the product expression was carried out by IPTG after the cell culture gained high density. The inducible expression product, due to the properties of the A2P peptide, was cleaved in the cell into SE-33-A2P peptides. As the next step, the SE-33-A2P peptide was purified using filtration and chromatography. Its activity against both Gram-positive and Gram-negative bacteria, including antibiotic-resistant bacteria, was proved. The developed approach for obtaining a prokaryotic system for the expression of a highly active antimicrobial peptide expands the opportunities for producing antimicrobial peptides via industrial methods

Overcoming the Limitations of Stem Cell-Derived Beta Cells

Great advances in type 1 diabetes (T1D) and type 2 diabetes (T2D) treatment have been made to this day. However, modern diabetes therapy based on insulin injections and cadaveric islets transplantation has many disadvantages. That is why researchers are developing new methods to regenerate the pancreatic hormone-producing cells in vitro. The most promising approach is the generation of stem cell-derived beta cells that could provide an unlimited source of insulin-secreting cells. Recent studies provide methods to produce beta-like cell clusters that display glucose-stimulated insulin secretion—one of the key characteristics of the beta cell. However, in comparison with native beta cells, stem cell-derived beta cells do not undergo full functional maturation. In this paper we review the development and current state of various protocols, consider advantages, and propose ways to improve them. We examine molecular pathways, epigenetic modifications, intracellular components, and the microenvironment as a possible leverage to promote beta cell functional maturation. A possibility to create islet organoids from stem cell-derived components, as well as their encapsulation and further transplantation, is also examined. We try to combine modern research on beta cells and their crosstalk to create a holistic overview of developing insulin-secreting systems

Transglutaminase 3: The Involvement in Epithelial Differentiation and Cancer

Transglutaminases (TGMs) contribute to the formation of rigid, insoluble macromolecular complexes, which are essential for the epidermis and hair follicles to perform protective and barrier functions against the environment. During differentiation, epidermal keratinocytes undergo structural alterations being transformed into cornified cells, which constitute a highly tough outermost layer of the epidermis, the stratum corneum. Similar processes occur during the hardening of the hair follicle and the hair shaft, which is provided by the enzymatic cross-linking of the structural proteins and keratin intermediate filaments. TGM3, also known as epidermal TGM, is one of the pivotal enzymes responsible for the formation of protein polymers in the epidermis and the hair follicle. Numerous studies have shown that TGM3 is extensively involved in epidermal and hair follicle physiology and pathology. However, the roles of TGM3, its substrates, and its importance for the integument system are not fully understood. Here, we summarize the main advances that have recently been achieved in TGM3 analyses in skin and hair follicle biology and also in understanding the functional role of TGM3 in human tumor pathology as well as the reliability of its prognostic clinical usage as a cancer diagnosis biomarker. This review also focuses on human and murine hair follicle abnormalities connected with TGM3 mutations

Early Stages of we/we wal/wal Mouse Hair Morphogenesis: Light and Fluorescent Microscopy of the Whole-Mount Epidermis

In adult skin, hair follicles cyclically self-renew in a manner that recapitulates embryonic hair follicle morphogenesis. The most common pathology of hair in adults is alopecia, which is hair loss to different extent. There are a number of murine models of alopecia including spontaneous mutations. In the present study, we worked with double homozygous we/we wal/wal mice which demonstrate symptoms closely resembling human alopecia. Using whole-mount preparations of epidermis of E18.5 embryos we show that hair follicle defects can be revealed as early as during embryonic morphogenesis in these mutants. The number of hair follicles was reduced almost 1.5-fold in mutant skin. The shape of the early stage small follicles was altered in mutant animals as compared to control ones. Additionally, follicles of mutant embryos were wider at the point of conjunction with interfollicular epidermis. We believe that the mutant mice studied represent a fascinating model to address the problem of hair loss. We demonstrated alterations in the morphogenesis of embryonic hair follicle in we/we wal/wal double homozygous mice developing alopecia postnatally. We suppose that incorrect morphogenesis of hair follicles during embryogenesis is closely related to alopecia in the adult life. Unveiling the mechanisms involved in altered embryogenesis may elucidate the pathogenesis of alopecia

Tissue-engineered biological dressing accelerates skin wound healing in mice via formation of provisional connective tissue

Despite recent advances in bioengineered therapies, wound healing remains a serious clinical problem. In acute full-thickness wounds, it is desirable to replace both the damaged dermis and epidermis in a single procedure. This approach requires appropriate properties of tissue-engineered dressings to support simultaneous regenerative processes in the dermis and epidermis while they are temporally separated in the natural wound healing process. In this study, a collagen-based scaffold inhabited by skin cells was employed. Its ability to stimulate the skin repair of full-thickness excisional splinting wounds in a murine model was evaluated in comparison with that of acellular collagen and commercially available gelatin porous sponge Spongostan®. The study showed that cell-based skin equivalent promoted the immediate filling of the wound bed and provided simultaneous reorganization of the dermal component into highly vascularized granulation-like tissue and rapid epithelialization, thus improving the quality of healing. Inflammation was delayed and less pronounced. In contrast, acellular collagen and especially Spongostan® failed to demonstrate similar results. The porous structure of Spongostan® prevented effective long-term epithelialization and impeded the formation of an adequate connective tissue at the wound bed

Trajectory of hiPSCs derived neural progenitor cells differentiation into dermal papilla-like cells and their characteristics

Abstract Dermal papilla cells (DPCs) play roles in key functions of the epidermis such as hair generation. The use of human induced pluripotent cells (hiPSCs) makes it possible to obtain DP-like cells and study the molecular mechanisms of DPC development during embryogenesis. In this work, we studied the phenotypic trajectory of hiPSCs during their differentiation into DP-like cells and evaluated the epithelial-mesenchymal interaction potential of the resulting cell line. Specifically, we differentiated hiPSCs into neural progenitor cells (NPCs) and subsequently into DP-like cells. Analysis of bulk RNA-seq data during this process enabled us to observe gene expression dynamics during five stages of dermal differentiation. Furthermore, functional assays (organoids in both collagen gels and hanging drop cultures and tubulogenesis assays) revealed that the dermal cell lines we generated could interact with epidermal cells

Immortalization Reversibility in the Context of Cell Therapy Biosafety

Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines