Blank Spots in the Map of Human Skin: The Challenge for Xenotransplantation.

Most of the knowledge about human skin homeostasis, development, wound healing, and diseases has been accumulated from human skin biopsy analysis by transferring from animal models and using different culture systems. Human-to-mouse xenografting is one of the fundamental approaches that allows the skin to be studied in vivo and evaluate the ongoing physiological processes in real time. Humanized animals permit the actual techniques for tracing cell fate, clonal analysis, genetic modifications, and drug discovery that could never be employed in humans. This review recapitulates the novel facts about mouse skin self-renewing, regeneration, and pathology, raises issues regarding the gaps in our understanding of the same options in human skin, and postulates the challenges for human skin xenografting.

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.

A Kaleidoscope of Keratin Gene Expression and the Mosaic of Its Regulatory Mechanisms.

Keratins are a family of intermediate filament-forming proteins highly specific to epithelial cells. A combination of expressed keratin genes is a defining property of the epithelium belonging to a certain type, organ/tissue, cell differentiation potential, and at normal or pathological conditions. In a variety of processes such as differentiation and maturation, as well as during acute or chronic injury and malignant transformation, keratin expression undergoes switching: an initial keratin profile changes accordingly to changed cell functions and location within a tissue as well as other parameters of cellular phenotype and physiology. Tight control of keratin expression implies the presence of complex regulatory landscapes within the keratin gene loci. Here, we highlight patterns of keratin expression in different biological conditions and summarize disparate data on mechanisms controlling keratin expression at the level of genomic regulatory elements, transcription factors (TFs), and chromatin spatial structure.

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.

Alveologenesis: What Governs Secondary Septa Formation.

The simplification of alveoli leads to various lung pathologies such as bronchopulmonary dysplasia and emphysema. Deep insight into the process of emergence of the secondary septa during development and regeneration after pneumonectomy, and into the contribution of the drivers of alveologenesis and neo-alveolarization is required in an efficient search for therapeutic approaches. In this review, we describe the formation of the gas exchange units of the lung as a multifactorial process, which includes changes in the actomyosin cytoskeleton of alveocytes and myofibroblasts, elastogenesis, retinoic acid signaling, and the contribution of alveolar mesenchymal cells in secondary septation. Knowledge of the mechanistic context of alveologenesis remains incomplete. The characterization of the mechanisms that govern the emergence and depletion of αSMA will allow for an understanding of how the niche of fibroblasts is changing. Taking into account the intense studies that have been performed on the pool of lung mesenchymal cells, we present data on the typing of interstitial fibroblasts and their role in the formation and maintenance of alveoli. On the whole, when identifying cell subpopulations in lung mesenchyme, one has to consider the developmental context, the changing cellular functions, and the lability of gene signatures.

Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is a group of inherited keratinopathies that, in most cases, arise due to mutations in keratins and lead to intraepidermal ruptures. The cellular pathology of most EBS subtypes is associated with the fragility of the intermediate filament network, cytolysis of the basal layer of the epidermis, or attenuation of hemidesmosomal/desmosomal components. Mutations in keratins 5/14 or in other genes that encode associated proteins induce structural disarrangements of different strengths depending on their locations in the genes. Keratin aggregates display impaired dynamics of assembly and diminished solubility and appear to be the trigger for endoplasmic reticulum (ER) stress upon being phosphorylated by MAPKs. Global changes in cellular signaling mainly occur in cases of severe dominant EBS mutations. The spectrum of changes initiated by phosphorylation includes the inhibition of proteasome degradation, TNF-α signaling activation, deregulated proliferation, abnormal cell migration, and impaired adherence of keratinocytes. ER stress also leads to the release of proinflammatory danger-associated molecular pattern (DAMP) molecules, which enhance avalanche-like inflammation. Many instances of positive feedback in the course of cellular stress and the development of sterile inflammation led to systemic chronic inflammation in EBS. This highlights the role of keratin in the maintenance of epidermal and immune homeostasis.

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.

Regenerative Effects and Development Patterns of Solid Neural Tissue Grafts Located in Gelatin Hydrogel Conduit for Treatment of Peripheral Nerve Injury.

BACKGROUND: The regeneration of the peripheral nerves after injuries is still a challenging fundamental and clinical problem. The cell therapy and nerve guide conduit construction are promising modern approaches. Nowadays, different sources of cells for transplantation are available. But it is little known about the interaction between fetal central nervous system cells and peripheral nerve tissue. In this study, we analyzed the development of the fetal neocortex and spinal cord solid grafts injected into the gelatin hydrogel conduits and their effects on sciatic nerve regeneration after cut injury. METHODS: Frontal neocortex tissue was obtained from E19.5 and spinal cord tissue was obtained from E14.5 fetuses harvested from transgenic EGFP mice. The grafts were injected into the hydrogel conduits which were connected to the nerve stumps after cut injury. The recovery of motor function was estimated with walking track analysis at 2, 5, and 8 weeks after surgery. Then immunohistochemical study was performed. RESULTS: The histological examination showed that only fetal neocortex solid graft cells had survived after implantation. Immunostaining revealed that some of the transplanted cells expressed neural markers such as neurofilament protein and NeuN. But the cells mostly differentiated in glial lineage, which was confirmed with immunostaining for GFAP and S100ß. The walking-track analysis has shown that 8 weeks after surgery bioengineered conduit differed significantly from the control. CONCLUSIONS: We revealed that the hydrogel conduit is suitable for nerve re-growth and that the fetal neocortex grafted cells can survive and differentiate. Bioengineered conduit can stimulate functional recovery after the nerve injury.

A mosaic intragenic microduplication of LAMA1 and a constitutional 18p11.32 microduplication in a patient with keratosis pilaris and intellectual disability.

The application of array-based comparative genomic hybridization and next-generation sequencing has identified many chromosomal microdeletions and microduplications in patients with different pathological phenotypes. Different copy number variations are described within the short arm of chromosome 18 in patients with skin diseases. In particular, full or partial monosomy 18p has also been associated with keratosis pilaris. Here, for the first time, we report a young male patient with intellectual disability, diabetes mellitus (type I), and keratosis pilaris, who exhibited a de novo 45-kb microduplication of exons 4-22 of LAMA1, located at 18p11.31, and a 432-kb 18p11.32 microduplication of paternal origin containing the genes METTL4, NDC80, and CBX3P2 and exons 1-15 of the SMCHD1 gene. The microduplication of LAMA1 was identified in skin fibroblasts but not in lymphocytes, whereas the larger microduplication was present in both tissues. We propose LAMA1 as a novel candidate gene for keratosis pilaris. Although inherited from a healthy father, the 18p11.32 microduplication, which included relevant genes, could also contribute to phenotype manifestation.

Multimodal label-free imaging of living dermal equivalents including dermal papilla cells.

BACKGROUND: Despite the significant progress in the development of skin equivalents (SEs), the problem of noninvasively assessing the quality of the cell components and the collagen structure of living SEs both before and after transplantation remains. Undoubted preference is given to in vivo methods of noninvasive, label-free monitoring of the state of the SEs. Optical bioimaging methods, such as cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), and fluorescence lifetime imaging microscopy (FLIM), present particular advantages for the visualization of such SEs. METHODS: In this study, we simultaneously applied several visualization techniques for skin model examination. We investigated the structure and quality of dermal equivalents containing dermal papilla (DP) cells and dermal fibroblasts (FBs) using CP OCT, MPT, and FLIM. Both the energy metabolism of the cell components and the structuring of the collagen fibrils were addressed. RESULTS: Based on the data from the fluorescence lifetimes and the contributions of protein-bound NAD(P)H, a bias toward oxidative metabolism was indicated, for the first time, in both the DP cells and FBs on day 14 of SE cultivation. The CP OCT and MPT data also indicated that both DP cells and FBs structured the collagen gel in a similar manner. CONCLUSION: In this study, multimodal label-free imaging of the structure and quality of living dermal equivalents was implemented for the first time with the use CP OCT, MPT, and FLIM of NAD(P)H. Our data suggest that the combination of different imaging techniques provides an integrated approach to data acquisition regarding the structure and quality of dermal equivalents, minimizes the potential disadvantages of using a single method, and provides an ideal information profile for clinical and research applications.

Label retaining cells and cutaneous stem cells.

This is a comprehensive review on label retaining cells (LRC) in epidermal development and homeostasis. The precise in vivo identification and location of epidermal stem cells is a crucial issue in cutaneous biology. We discuss here the following problems: (1) Identification and location of LRC in the interfollicular epithelium and hair follicle; (2) The proliferative potential of LRC and their role in cutaneous homeostasis (3); LRC phenomenon and the Immortal Strand Hypothesis, which suggests an alternative mechanism for retention of genetic information; (4) Significance of LRC studies for development of stem cell concept. Now, it seems evident that LRC are a frequent feature of stem cell niches and revealing highly dormant LRC may be used for identification of stem cell niches in different tissues. LRC were used for screening specific markers of epidermal stem cells. Within a given tissue stem cells have different proliferative characteristics. There are more frequently cycling stem cells which function primarily in homeostasis, while LRC form a reserve of dormant, may be ultimate, stem cells, which are set aside for regeneration of injury or unforeseen need. The authors suggest that LRC dormancy described in Mammalia has much in common with developmental quiescence found in some other animals. For example in C. elegans reproductive system, vulval precursor cells have developmentally programmed cell-cycle arrest in the first larval stage, and then undergo an extended period of quiescence before resuming proliferation. Another example of developmental quiescence is the diapause, a widespread phenomenon exhibited by animals ranging from nematodes to mammals, often occurring at genetically predetermined life history stage.

Dermal papilla cells induce keratinocyte tubulogenesis in culture.

The ability of dermal papilla (DP) cells to induce hair growth was reported in many studies. However, early stages of hair follicle development and signals that govern this process are poorly understood. Therefore, an in vitro model may be a convenient system to study epithelial-mesenchymal interactions and early stages of epidermal morphogenesis, especially in humans. To investigate the role of DP cells in epidermal morphogenesis we modified the method of isolation of DP cells from hair follicle of human scalp and developed the three-dimensional model of epidermal morphogenesis. Isolated DP cells were able to differentiate in adipogenic and osteogenic directions and retained activity of alkaline phosphatase (AP) for seven passages in culture. DP cells were able to induce tubule-like structures in three-dimensional model in vitro and to reorganize collagen matrix. Prolonged cultivation of DP cells has been a big problem because of the loss of hair follicle-inducing ability and growth activity after several passages. To solve this problem we immortalized DP cells by the transfection of the human telomerase reverse transcriptase cDNA (hTERT). Immortalized DP-hTERT cells retained AP activity and demonstrated low ability to osteogenic differentiation. The conditioned medium collected from actively proliferated cells as well as DP-hTERT cells themselves were capable to induce tubulogenesis after prolonged keratinocyte cultivation.