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The development of technologies for the generation and transplantation of living skin equivalents (LSEs) is a significant area of translational medicine. Such functional equivalents can be used to model and study the morphogenesis of the skin and its derivatives, to test drugs, and to improve the healing of chronic wounds, burns, and other skin injuries. The evolution of LSEs over the past 50 years has demonstrated the leap in technology and quality and the shift from classical full-thickness LSEs to principled new models, including modification of classical models and skin organoids with skin derived from human-induced pluripotent stem cells (iPSCs) (hiPSCs). Modern methods and approaches make it possible to create LSEs that successfully mimic native skin, including derivatives such as hair follicles (HFs), sebaceous and sweat glands, blood vessels, melanocytes, and nerve cells. New technologies such as 3D and 4D bioprinting, microfluidic systems, and genetic modification enable achievement of new goals, cost reductions, and the scaled-up production of LSEs.
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Células Madre Pluripotentes Inducidas , Piel , Ingeniería de Tejidos , Humanos , Ingeniería de Tejidos/métodos , Células Madre Pluripotentes Inducidas/citología , Piel Artificial , Organoides , Modelos Biológicos , Bioimpresión/métodos , Cicatrización de Heridas/fisiologíaRESUMEN
Extensive skin damage requires specialized therapy that stimulates regeneration processes without scarring. The possibility of using combination of a collagen gel application as a wound dressing and fibroblast attractant with verteporfin as an antifibrotic agent was examined in vivo and in vitro. In vitro effects of verteporfin on viability and myofibroblast markers expression were evaluated using fibroblasts isolated from human scar tissue. In vivo the collagen gel and verteporfin (individually and in combination) were applied into the wound to investigate scarring during skin regeneration: deviations in skin layer thickness, collagen synthesis, and extracellular matrix fibers were characterized. The results indicate that verteporfin reduces fibrotic phenotype by suppressing expression of the contractile protein Sm22α without inducing cell death. However, administration of verteporfin in combination with the collagen gel disrupts its ability to direct wound healing in a scarless manner, which may be related to incompatibility of the mechanisms by which collagen and verteporfin control regeneration.
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Colágeno , Fibroblastos , Verteporfina , Verteporfina/farmacología , Verteporfina/uso terapéutico , Humanos , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Animales , Cicatrización de Heridas/efectos de los fármacos , Antifibróticos/farmacología , Antifibróticos/uso terapéutico , Células Cultivadas , Andamios del Tejido/química , Cicatriz/tratamiento farmacológico , Cicatriz/patología , Cicatriz/metabolismo , Masculino , Fibrosis , Piel/efectos de los fármacos , Piel/patología , Piel/metabolismoRESUMEN
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.
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With the development of regenerative medicine in ophthalmology, the identification of cells with high proliferative potential in the limbal area has attracted the attention of ophthalmologists and offered a new option for treatment in clinical practice. Limbal stem cell deficiency (LSCD) is an identified eye disease with a difficult and negative outcome, for which the traditional treatment is keratoplasty. This study sought to evaluate the efficacy of matrix-assisted cell transplantation consisting of in vitro-cultured autologous limbal stem cells (LSCs) and type I collagen for the treatment of LSCD in rabbits. LSCD was induced in 10 rabbits by a combination of mechanical limbectomy and alkali burns. Cells were cultured on a plate for 14 days before being transferred to a collagen-based matrix for another 7 days. Rabbits were divided into two groups as follows: the experimental group (five rabbits) received matrix-assisted cell transplantation, while the control group (five rabbits) received only conservative therapy with anti-inflammatory eye drops. During the postoperative period, all rabbits were examined using slit-lamp biomicroscopy with photo-registration and fluorescent staining, impression cytology and anterior segment optical coherence tomography (AS-OCT). Rabbits were euthanized at 30 and 120 days, and their corneas were processed for histology and immunohistochemistry. As a consequence, rabbits in the experimental group demonstrated the restoration of the corneal epithelium and transparency without epithelial defects. Moreover, goblet cells were absent in the central zone of the corneal epithelium. In conclusion, our new method of treatment enhanced the corneal surface and is an effective method of treatment for LSCD in rabbits.
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Biodiversity collections are important vehicles for protecting endangered wildlife in situations of adverse anthropogenic influence. In Russia, there are currently a number of institution- and museum-based biological collections, but there are no nation-wide centres of biodiversity collections. In this paper, we report on the results of our survey of 324 bioconservation, big-data, and ecology specialists from different regions of Russia in regard to the necessity to create several large national biodiversity centres of wildlife protection. The survey revealed specific goals that have to be fulfilled during the development of these centres for the protection and restoration of endangered wildlife species. The top three problems/tasks (topics) are the following: (1) the necessity to create large national centres for different types of specimens; (2) the full sequencing and creation of different "omic" (genomic, proteomic, transcriptomic, etc.) databases; (3) full digitisation of a biodiversity collection/centre. These goals may constitute a guideline for the future of biodiversity collections in Russia that would be targeted at protecting and restoring endangered species. With the due network service level, the translation of the website into English, and permission from the regulator (Ministry of Science and Higher Education of Russian Federation), it can also become an international project.
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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.
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Piel , Cicatrización de Heridas , Humanos , Animales , Ratones , Trasplante Heterólogo , Xenoinjertos , BiopsiaRESUMEN
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.
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Células Epidérmicas , Células-Madre Neurales , Humanos , Diferenciación Celular , Organoides , BioensayoRESUMEN
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.
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Contención de Riesgos Biológicos , Telomerasa , Línea Celular , Diferenciación Celular , Proliferación Celular , Telomerasa/metabolismoRESUMEN
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.
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Células Epiteliales , Queratinas , Queratinas/genética , Queratinas/metabolismo , Epitelio/metabolismo , Células Epiteliales/metabolismo , Citoesqueleto/metabolismo , Expresión GénicaRESUMEN
iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Fluorescente , NAD/metabolismoRESUMEN
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.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Diferenciación Celular , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Células Madre/metabolismoRESUMEN
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.
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Actomiosina/genética , Pulmón/crecimiento & desarrollo , Organogénesis/genética , Alveolos Pulmonares/crecimiento & desarrollo , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patología , Linaje de la Célula/genética , Citoesqueleto/genética , Enfisema/genética , Enfisema/patología , Gases/metabolismo , Humanos , Pulmón/patología , Mesodermo/citología , Mesodermo/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Tretinoina/metabolismoRESUMEN
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.
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Alarminas/genética , Epidermis/metabolismo , Epidermólisis Ampollosa Simple/genética , Queratina-14/genética , Queratina-5/genética , Queratinocitos/metabolismo , Alarminas/metabolismo , Estrés del Retículo Endoplásmico/genética , Epidermis/patología , Epidermólisis Ampollosa Simple/metabolismo , Epidermólisis Ampollosa Simple/patología , Regulación de la Expresión Génica , Humanos , Inflamación , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Filamentos Intermedios/ultraestructura , Queratina-14/metabolismo , Queratina-5/metabolismo , Queratinocitos/patología , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Proteolisis , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.
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Fibroblastos/metabolismo , Recombinación Homóloga , Integrasas/metabolismo , Queratinocitos/metabolismo , Telomerasa/genética , Transgenes , Adolescente , Adulto , Biomarcadores , Línea Celular Transformada , Proliferación Celular , Senescencia Celular/genética , Niño , Epidermólisis Ampollosa Distrófica/etiología , Epidermólisis Ampollosa Distrófica/metabolismo , Femenino , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Orden Génico , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Cultivo Primario de Células , Proteómica/métodos , Telomerasa/metabolismo , Adulto JovenRESUMEN
The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.
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Colágeno Tipo VII , Dermis , Epidermólisis Ampollosa Distrófica , Fibroblastos , Regulación de la Expresión Génica , Homocigoto , Mutación , Adolescente , Adulto , Niño , Colágeno Tipo VII/biosíntesis , Colágeno Tipo VII/genética , Dermis/metabolismo , Dermis/patología , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs.
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Diferenciación Celular , Cuerpos Embrioides/citología , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Células Madre Pluripotentes Inducidas/citología , Meiosis , Células Cultivadas , Cuerpos Embrioides/metabolismo , Femenino , Perfilación de la Expresión Génica , Células Germinativas/metabolismo , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/metabolismo , RNA-SeqRESUMEN
There are many studies devoted to the role of hair follicle stem cells in wound healing as well as in follicle self-restoration. At the same time, the influence of the inflammatory cells on the hair follicle cycling in both injured and intact skin is well established. Immune cells of all wound healing stages, including macrophages, γδT cells, and T regs, may activate epidermal stem cells to provide re-epithelization and wound-induced hair follicle neogenesis. In addition to the ability of epidermal cells to maintain epidermal morphogenesis through differentiation program, they can undergo de-differentiation and acquire stem features under the influence of inflammatory milieu. Simultaneously, a stem cell compartment may undergo re-programming to adopt another fate. The proportion of skin resident immune cells and wound-attracted inflammatory cells (e.g., neutrophils and macrophages) in wound-induced hair follicle anagen and plucking-induced anagen is still under discussion to date. Experimental data suggesting the role of reactive oxygen species and prostaglandins, which are uncharacteristic of the intact skin, in the hair follicle cycling indicates the role of neutrophils in injury-induced conditions. In this review, we discuss some of the hair follicles stem cell activities, such as wound-induced hair follicle neogenesis, hair follicle cycling, and re-epithelization, through the prism of inflammation. The plasticity of epidermal stem cells under the influence of inflammatory microenvironment is considered. The relationship between inflammation, scarring, and follicle neogenesis as an indicator of complete wound healing is also highlighted. Taking into consideration the available data, we also conclude that there may exist a presumptive interlink between the stem cell activation, inflammation and the components of programmed cell death pathways.
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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.
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Células Epiteliales/metabolismo , Neoplasias/genética , Transglutaminasas/metabolismo , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
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.
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Modeling organoids with hair follicle germ-like properties provides an opportunity for developing strategies for alopecia drug discovery and replacement therapy, as well as investigating the molecular mechanisms underlying human hair follicle regeneration in vitro. Hair follicle germ reconstruction in vitro is based on dermal papilla hair-inducing abilities and the plasticity of skin epidermal keratinocytes. The current protocol describes a highly efficient approach suitable for adult human skin cell applications. This method allows to obtain hair follicle germs using tissues from one donor. Isolated and cultured for 2 weeks, adult hair follicle dermal papilla cells and skin epidermal keratinocytes self-organize in hanging drop cultures generating organoids that exhibit the features of folliculogenesis onset.