A new stable line of human keratinocytes was obtained. The cells have altered morphology, both abnormal chromosomal composition and expression of keratinocyte markers, do not show contact inhibition, could be cultured in various media and have limited stratification ability in vitro. Upon transplantation into nude mice the cells have tumorigenic properties.

Xenotransplantation of skin transplants from humans to animals with immunodeficiency is one of the most successful models for studying human tissues in normal and pathological conditions. The main limitation of this approach is the size of the graft; the lower part of the dermis and subcutaneous fat is missing in skin explants with an area of more than 10 × 10 mm, and full-layer xenografts rarely exceed the size of 3 × 3 mm. This study shows that transplantation of 15 × 5 mm full-layer strips ensures successful integration and preservation of all skin structures: the epidermis, dermal part, blood vessels, hair follicles, and sebaceous and sweat glands. Hair follicles in the grafts reproduce the cycle of physiological regeneration. This model is suitable for studying the regeneration of hair follicles, glands, interfollicular epidermis, and other skin structures.

De novo hair follicle (HF) formation in embryonic skin and hair growth in postnatal skin are the result of epithelial-mesenchymal interactions between specialized mesenchymal dermal papilla (DP) and epithelial stem cells that give rise to hairs. Adult HF is a valuable source of diferent lineages of stem cells (SCs) with morphogenetic potential. Epithelial stem cells are residing in the special compartment of HF (the bulge) and can be mobilized to regenerate the new follicle with each hair cycle and to reepithelialize epidermis during wound repair. This review summarizes the current knowledge on key characteristics of HF SC populations in terms of regenerative potential. General biological principles that govern the mesenchymal-epithelial interactions within the HF and the signaling pathways that control HF development are discussed. The main focus is on recent approaches to reconstruct folliculogenesis in vitro and perspectives of the tissue engineering in alopecia therapy.

Possible ways to extend cultivation of dermal papilla cells without the loss of expression of their specific markers were studied. The effect of extracellular matrix components, as well as valproic acid, on the maintenance of the phenotype of dermal papilla cells was studied for the first time. Two ways of cultivation (in a monolayer culture and in spheroids) were used. It was established that a short-term positive effect is reached during the addition of the BMP6 growth factor and vitamin D3 in the monolayer culture, while cultivation in spheroids or in the presence of valproic acid allows us to preserve most efficiently the initial phenotype of these cells in vitro. The significance of the results obtained for tissue engineering and for the study of regeneration processes is discussed.

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.

De novo hair follicle (HF) formation in embryonic skin and hair growth in postnatal skin are the result of epithelial-mesenchymal interactions between specialized mesenchymal dermal papilla (DP) and epithelial stem cells that give rise to hairs. Adult HF is a valuable source of different lineages of stem cells (SCs) with morphogenetic potential. Epithelial stem cells are residing in the special compartment of HF (the bulge) and can be mobilized to regenerate the new follicle with each hair cycle and to reepithelialize epidermis during wound repair. This review summarizes the current knowledge on key characteristics of HF SC populations in terms of regenerative potential. General biological principles that govern the mesenchymal-epithelial interactions within the HF and the signaling pathways that control HF development are discussed. The main focus is on recent approaches to reconstruct folliculogenesis in vitro and perspectives of the tissue engineering in alopecia therapy.

Dopamine in the concentration 0.4 μg/mL abolishes protein synthesis rhythm in HaCaT keratinocytes and hepatocytes unlike noradrenaline or melatonin, which synchronize direct intercellular interactions and organize protein synthesis rhythm. Experiments with D2 dopamine receptors blocking agent metoclopramide (tserukal) in the concentration 2 μg/mL show that a disorganizing effect of dopamine is driven by the activation of D2 receptors, which block adenylyl cyclase and the efflux of calcium ions from internal depos according to the literature. It is shown that tserukal does not activate serotonin receptors in our experimental settings. Cellular interactions’ recovery during or after dopamine action is carried out by melatonin in the concentration 0.001 μg/mL. A recommendation to inject melatonin before dopamine administration for different medical indications is discussed.

The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

Изучение фенотипической пластичности и дифференцировочных способностей клеток является важной задачей клеточной биологии. В представленной работе исследована способность клеток поднижнечелюстной слюнной железы мыши к дифференцировке в гепатоцитарном направлении, а также проанализировано влияние вальпроевой кислоты, ингибитора гистон-деацетилаз, на эффективность дифференцировки. Показано, что после дифференцировки в клетках слюнной железы возрастает экспрессия генов гепатоцитарных маркеров (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) и факторов транскрипции (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6). Вальпроевая кислота увеличивает специфичность гепатоцитарной дифференцировки, снижая экспрессию протоковых (Krt19, Hhex1, Cyp7a1) и ацинарных (Ptf1a) маркеров. После воздействия вальпроевой кислоты усиливается также эффективность гепатоцитарной дифференцировки, о чем говорит повышение уровня Alb и Tdo2 и увеличение секреции мочевины дифференцированными клетками. Изменений в характере метилирования промоторных областей генов не обнаружено, однако обработка вальпроевой кислотой и гепатоцитарная дифференцировка в значительной степени затрагивают метилирование гистона H3, ассоциированного с генами, ключевыми для гепатоцитарной дифференцировки. Таким образом, клетки поднижнечелюстной слюнной железы мыши способны к дифференцировке в гепатоцитарном направлении, а вальпроевая кислота увеличивает специфичность и эффективность их дифференцировки.

Mice with skin and hair follicle (HF) defects are common models of human skin disorders. A mutant strain with the we/we wal/wal genotype develops alopecia. We found the hair shaft structure in the pelage of mutant mice to have significant defects. Although these mice lose their hair at 21 days, a label-retaining cell population persists in HFs until at least day 54. Depilation-induced anagen was accomplished in we/we wal/wal mutants but the resulting hair shafts were short and extremely deformed. Serious abnormalities in epidermis stratification and HF morphogenesis exist in we/we wal/wal homozygous E18.5 embryos. There were significantly fewer HF primordia in this mutant compared with wild type. We discovered specific structures, identified as invalid placodes, positive for ectodysplasin A1 receptor, nuclear β-catenin, and LEF1, which failed to invaginate, produced a double basal-like layer of epidermal cells, and lacked cylindrical keratinocytes. Specification of dermal papillae (DP) was impaired, and the papillary dermis expressed alkaline phosphatase and LEF1. We also detected DP-like groups of intensively stained cells in the absence of visible signs of folliculogenesis in the epidermis. We showed differentiation disturbances in the mutant embryonic E18.5 epidermis and HFs: The cornified layer was absent, the width of the spinous layer was reduced, and HFs lacked LEF1-positive precortex cells. In this study, we used a very interesting and useful mouse model of alopecia. The presence of symptoms of skin disorders in we/we wal/wal murine embryos correlates with the postnatal skin phenotype. This correlation may help to evaluate reasons of alopecia.

We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3.

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.

Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.