Search

Basal cell carcinoma (BCC) of the skin, the most common malignancy in individuals of mixed European descent, is increasing in incidence due to an aging population and sun exposure habits. The realization that aberrant activation of Hedgehog signaling is a pathognomonic feature of BCC development has opened the way for exciting progress toward understanding BCC biology and translation of this knowledge to the clinic. Genetic mouse models closely mimicking human BCCs have provided answers about the tumor cell of origin, and inhibition of Hedgehog signaling is emerging as a potentially useful targeted therapy for patients with advanced or multiple BCCs that have hitherto lacked effective treatment. [ABSTRACT FROM AUTHOR]

The article discusses research being done on the self-renewal of small intestine tissue, colon and hair follicles from cycling Lgr5+ve stem cells in adult mammals. The researchers discovered that the Lgr5+ve cells of the small intestine appear to be more self-renewing than colon. They believe that the Lgr5+ve cells of the three organs studied constitute stem cell pools that persist for the lifetime of the mouse used in the research.

Common characteristics in the pathogenesis of psoriasis (PS) and atopic dermatitis (AD) have been presumed, but only a few studies have clearly supported this. The current aim was to find possible similarities and differences in protein expression patterns between these two major chronic inflammatory skin diseases. High-throughput tandem mass spectrometry proteomic analysis was performed using full thickness skin samples from adult PS patients, AD patients and healthy subjects. We detected a combined total of 3045 proteins in the three study groups. According to principal component analysis, there was significant overlap between the proteomic profiles of PS and AD, and both clearly differed from that of healthy skin. The following validation of selected proteins with western blot analysis showed similar tendencies in expression levels and produced statistically significant results. The expression of periostin (POSTN) was consistently high in AD and very low or undetectable in PS (5% FDR corrected p < 0.001), suggesting POSTN as a potential biomarker to distinguish these diseases. Immunohistochemistry further confirmed higher POSTN expression in AD compared to PS skin. Overall, our findings support the concept that these two chronic skin diseases might share considerably more common mechanisms in pathogenesis than has been suspected thus far., (© 2024 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

As in other mammalian tissues, the extracellular matrix (ECM) of skin functions as mechanical support and regulative environment that guides the behavior of the cells. ECM is a gel-like structure that is primarily composed of structural and nonstructural proteins. While the content of structural proteins is stable, the level of nonstructural ECM proteins, such as thrombospondin-4 (THBS4), is dynamically regulated. In a previous work we demonstrated that THBS4 stimulated cutaneous wound healing. In this work we discovered that in addition to proliferation, THBS4 stimulated the migration of primary keratinocytes in 3D. By using a proteotransciptomic approach we found that stimulation of keratinocytes with THBS4 regulated the activity of signaling pathways linked to proliferation, migration, inflammation and differentiation. Interestingly, some of the regulated genes (eg IL37, TSLP) have been associated with the pathogenesis of atopic dermatitis (AD). We concluded that THBS4 is a promising candidate for novel wound healing therapies and suggest that there is a potential convergence of pathways that stimulate cutaneous wound healing with those active in the pathogenesis of inflammatory skin diseases., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Lichen planus is a chronic inflammatory mucocutaneous disease that belongs to the group of papulosquamous skin diseases among diseases like psoriasis, a widely studied disease in dermatology. The aim of the study was to identify the changes between the blood sera of lichen planus patients and healthy controls to widen the knowledge about the metabolomic aspect of lichen planus and gain a better understanding about the pathophysiology of the disease. We used high-throughput nuclear magnetic resonance (NMR) spectroscopy to measure the levels of blood serum metabolites, lipoproteins and lipoprotein particles. Dyslipidemia has relatively recently been shown to be one of the comorbidities of lichen planus, but the changes in the components of lipoproteins have not been described yet. We found statistically significant changes in the concentrations of 16 markers regarding lipoproteins, which included the components of intermediate-density lipoproteins, low-density lipoproteins and large low-density lipoproteins. We propose that the detected changes may increase the risk for specific comorbidities (e.g., dyslipidemia) and resulting cardiovascular diseases, as the turnover and hepatic uptake of the altered/modified lipoprotein particles are disturbed.

Dupuytren's contracture (DC) is a chronic and progressive fibroproliferative disorder restricted to the palmar fascia of the hands. Previously, we discovered the presence of high levels of connective tissue growth factor in sweat glands in the vicinity of DC nodules and hypothesized that sweat glands have an important role in the formation of DC lesions. Here, we shed light on the role of sweat glands in the DC pathogenesis by proteomic analysis and immunofluorescence microscopy. We demonstrated that a fraction of sweat gland epithelium underwent epithelial-mesenchymal transition illustrated by negative regulation of E-cadherin. We hypothesized that the increase in connective tissue growth factor expression in DC sweat glands has both autocrine and paracrine effects in sustaining the DC formation and inducing pathological changes in DC-associated sweat glands.

Cutaneous wound healing is a complex process that encompasses alterations in all aspects of the skin including the extracellular matrix (ECM). ECM consist of large structural proteins such as collagens and elastin as well as smaller proteins with mainly regulative properties called matricellular proteins. Matricellular proteins bind to structural proteins and their functions include but are not limited to interaction with cell surface receptors, cytokines, or protease and evoking a cellular response. The signaling initiated by matricellular proteins modulates differentiation and proliferation of cells having an impact on the tissue regeneration. In this review we give an overview of the matricellular proteins that have been found to be involved in cutaneous wound healing and summarize the information known to date about their functions in this process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cárdenas-León, Mäemets-Allas, Klaas, Lagus, Kankuri and Jaks.)

Atopic dermatitis (AD) and psoriasis (PS) are common chronic inflammatory dermatoses. Although the differences at the intercellular and intracellular signaling level between AD and PS are well described, the resulting differences at the metabolism level have not yet been systematically analyzed. We compared the metabolomic profiles of the lesional skin, non-lesional skin and blood sera of AD and PS. Skin biopsies from 15 patients with AD, 20 patients with PS and 17 controls were collected, and 25 patients with AD, 55 patients with PS and 63 controls were recruited for the blood serum analysis. Serum and skin samples were analyzed using a targeted approach to find the concentrations of 188 metabolites and their ratios. A total of 19 metabolites differed in the comparison of lesional skins, one metabolite in non-lesional skins and 5 metabolites in blood sera. Although we found several metabolomic similarities between PS and AD, clear differences were outlined. Sphingomyelins were elevated in lesional skin of AD, implying a deficient barrier function. Increased levels of phosphatidylcholines, carnitines and asymmetric dimethylarginine in PS lesional skin and carnitines amino acids in the PS serum pointed to elevated cell proliferation. The comparison of the metabolomic profiles of AD and PS skin and sera outlined distinct patterns that were well correlated with the differences in the pathogenetic mechanisms of these two chronic inflammatory dermatoses.

Olfactomedin 4 (OLFM4), a multifunctional matricellular protein, is involved in regulation of angiogenesis, innate immunity, inflammation, tumorigenesis and metastasis formation via modulation of important cellular processes like adhesion, proliferation, differentiation as well as apoptosis. In our previous work we demonstrated the upregulation of OLFM4 during liver regeneration and cutaneous wound healing. Here we studied the outcomes of OLFM4 downregulation in human immortalized keratinocytes - the HaCaT cells. The suppression of OLFM4 inhibited migration but enhanced the proliferation of these cells. By using proteomic, and phosphoproteomic analysis, we found that OLFM4 downregulation induced changes in the levels of 184 proteins and 348 phosphosites. An integrated pathway analysis suggested that the increased phosphorylation of CDK7 at Ser164 and Thr170 may serve as the key event in the activation of CDK2 and consequent activation of cell cycle progression. Furthermore, the decrease in GIT1 and WAVE2 protein levels were connected to the disorganization of the actin cytoskeleton, reduction of lamellipodia formation at the leading edge of HaCaT cells, and decrease in their migration capacity., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Olfactomedin-4 (OLFM4) is an olfactomedin-domain-containing glycoprotein, which regulates cell adhesion, proliferation, gastrointestinal inflammation, innate immunity and cancer metastasis. In the present study we investigated its role in skin regeneration. We found that OLFM4 expression is transiently upregulated in the proliferative phase of cutaneous wound healing in humans as well as in mice. Moreover, a significant increase in OLFM4 expression was detected in the skin of lesional psoriasis, a chronic inflammatory disease characterized by keratinocyte hyperproliferation. In vitro experiments demonstrated that OLFM4 selectively stimulated keratinocyte proliferation and increased both keratinocyte and fibroblast migration. Using proteotranscriptomic pathway analysis we revealed that transcription factors POU5F1/OCT4 and ESR1 acted as hubs for OLFM4-induced signalling in keratinocytes. In vivo experiments utilizing mouse splinted full-thickness cutaneous wound healing model showed that application of recombinant OLFM4 protein can significantly improve wound healing efficacy. Taken together, our results suggest that OLFM4 acts as a transiently upregulated inflammatory signal that promotes wound healing by regulating both dermal and epidermal cell compartments of the skin., (© 2022. The Author(s).)

Solution blow spinning (SBS) has recently emerged as a novel method that can produce nano- and microfiber structures suitable for tissue engineering. Gelatin is an excellent precursor for SBS as it is derived mainly from collagens that are abundant in natural extracellular matrices. Here we report, for the first time the successful generation of 3D thermally crosslinked preforms by using SBS from porcine gelatin. These SBS mats were shown to have three-dimensional fibrous porous structure similar to that of mammalian tissue extracellular matrix. In pharma industry, there is an urgent need for adequate 3D liver tissue models that could be used in high throughput setting for drug screening and to assess drug induced liver injury. We used SBS mats as culturing substrates for human hepatocytes to create an array of 3D human liver tissue equivalents in 96-well format. The SBS mats were highly cytocompatible, facilitated the induction of hepatocyte specific CYP gene expression in response to common medications, and supported the maintenance of hepatocyte differentiation and polarization status in long term cultures for more than 3 weeks. Together, our results show that SBS-generated gelatin scaffolds are a simple and efficient platform for use in vitro for drug testing applications., (© 2021. The Author(s).)

Thrombospondin-4 (THBS4) is a non-structural extracellular matrix molecule associated with tissue regeneration and a variety of pathological processes characterized by increased cell proliferation and migration. However, the mechanisms of how THBS4 regulates cell behavior as well as the pathways contributing to its effects have remained largely unexplored. In the present study we investigated the role of THBS4 in skin regeneration both in vitro and in vivo . We found that THBS4 expression was upregulated in the dermal compartment of healing skin wounds in humans as well as in mice. Application of recombinant THBS4 protein promoted cutaneous wound healing in mice and selectively stimulated migration of primary fibroblasts as well as proliferation of keratinocytes in vitro . By using a combined proteotranscriptomic pathway analysis approach we discovered that β-catenin acted as a hub for THBS4-dependent cell signaling and likely plays a key role in promoting its downstream effects. Our results suggest that THBS4 is an important contributor to wound healing and its incorporation into novel wound healing therapies may be a promising strategy for treatment of cutaneous wounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klaas, Mäemets-Allas, Heinmäe, Lagus, Cárdenas-León, Arak, Eller, Kingo, Kankuri and Jaks.)

We describe a case of Lichtenberg Figures (LFs) following an electrical injury from a high-voltage switchgear in a 47 year-old electrician. LFs, also known as ferning pattern or keraunographic markings, are a pathognomonic skin sign for lightning strike injuries. Their true pathophysiology has remained a mystery and only once before described following an electical injury. The aim was to characterise the tissue response of LFs by performing untargeted non-labelled proteomics and immunohistochemistry on paraffin-embedded sections of skin biopsies taken from the area of LFs at presentation and at 3 months follow-up. Our results demonstrated an increase in dermal T-cells and greatly increased expression of the iron-binding glycoprotein lactoferrin by keratinocytes and lymphocytes. These changes in the LF-affected skin were associated with extravasation of red blood cells from dermal vessels. Our results provide an initial molecular and cellular insight into the tissue response associated with LFs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lindford, Juteau, Jaks, Klaas, Lagus, Vuola and Kankuri.)

Primary cilia (PC) are solitary, post-mitotic, microtubule-based, and membrane-covered protrusions that are found on almost every mammalian cell. PC are specialized cellular sensory organelles that transmit environmental information to the cell. Signaling through PC is involved in the regulation of a variety of cellular processes, including proliferation, differentiation, and migration. Conversely, defective, or abnormal PC signaling can contribute to the development of various pathological conditions. Our knowledge of the role of PC in organ development and function is largely based on ciliopathies, a family of genetic disorders with mutations affecting the structure and function of PC. In this review, we focus on the role of PC in their major signaling pathways active in skin cells, and their contribution to wound healing and scarring. To provide comprehensive insights into the current understanding of PC functions, we have collected data available in the literature, including evidence across cell types, tissues, and animal species. We conclude that PC are underappreciated subcellular organelles that significantly contribute to both physiological and pathological processes of the skin development and wound healing. Thus, PC assembly and disassembly and PC signaling may serve as attractive targets for antifibrotic and antiscarring therapies., (Copyright © 2020 Hosio, Jaks, Lagus, Vuola, Ogawa and Kankuri.)

Human mesenchymal stromal cells (MSC) are an important tool for basic and translational research. Large amounts of MSC are required for in vitro and in vivo studies, however, the limited life-span and differentiation ability in vitro hamper their optimal use. Here we report that 1:1 mixture of L15 and mTeSR1 culture media increased the life-span of IPI-SA3-C4, a normal non-immortalized human subcutaneous preadipocyte strain by 20% while retaining their adipogenic capacity and stable karyotype. The increased proliferative capacity was accompanied by increased expression of the stem markers POU5F1, SOX2, MYC and hTERT, and inhibition of hTERT activity abolished the growth advantage of L15-mTeSR1. Consequently, the described MSC culture would considerably enhance the utility of MSC for in vitro studies., Competing Interests: Declaration of competing interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Murine 3T3 cell lines constitute a standard model system for in vitro study of mammalian adipogenesis although they do not faithfully reflect the biology of the human adipose cells. Several human adipose cell lines and strains have been used to recapitulate human adipogenesis in vitro, but to date there is no generally accepted in vitro model for human adipogenesis. We obtained a clonal strain of human subcutaneous adipose stromal cells, IPI-SA3-C4, and characterized its utility as an in vitro model for human subcutaneous adipogenesis. IPI-SA3-C4 cells showed a high proliferative potential for at least 30 serial passages, reached 70 cumulative population doublings and exhibited a population doubling time of 47 h and colony forming efficiency of 12% at the 57th cumulative population doublings. IPI-SA3-C4 cells remained diploid (46XY) even at the 56th cumulative population doublings and expressed the pluripotency markers POU5F1, NANOG, KLF4, and MYC even at 50th cumulative population doublings. Under specific culture conditions, IPI-SA3-C4 cells displayed cellular hallmarks and molecular markers of adipogenic, osteogenic, and chondrogenic lineages and showed adipogenic capacity even at the 66th cumulative population doublings. These characteristics show IPI-SA3-C4 cells as a promising potential model for human subcutaneous adipogenesis in vitro.

Lgr5-LacZ mice harbor the Escherichia coli LacZ gene encoding β-galactosidase (β-gal) under the control of the Lgr5 promoter, a stem/progenitor cell marker. In injured livers of Lgr5-LacZ mice, cells expressing β-galactosidase (β-gal) are considered as potential bipotent liver progenitors; however, their origin and identity remain unknown. Unexpectedly, using lineage tracing, we demonstrate that the β-gal + cells do not originate from liver parenchymal cells. Instead, β-gal + cells, isolated from injured livers of both Lgr5-LacZ and wild-type mice, are positive for markers of Kupffer cells, liver-resident macrophages. The β-gal expression in these cells is a result of elevated expression of the endogenous beta-galactosidase Glb1. In injured livers of Lgr5-LacZ mice, bacterial β-gal expression is very low, suggesting transgene silencing. The gene expression profile of the β-gal + Kupffer cells from injured livers suggests a role in liver regeneration., (© 2019 Federation of European Biochemical Societies.)

Systematic understanding of the metabolite signature of diseases may lead to a closer understanding of the disease pathogenesis and ultimately to the development of novel therapies and diagnostic tools. Here we compared for the first time the full metabolomic profiles of lesional and non-lesional skin biopsies obtained from plaque psoriasis patients and skin samples of healthy controls. Significant differences in the concentration levels of 29 metabolites were identified that provide several novel insights into the metabolic pathways of psoriatic lesions. The metabolomic profile of the lesional psoriatic skin is mainly characterized by hallmarks of increased cell proliferation. As no significant differences were identified between non-lesional skin and healthy controls we conclude that local inflammatory process that drives the increased cell proliferation is the main cause of the identified metabolomic shifts.

Because molecular memories of past inflammatory events can persist in epidermal cells, we evaluated the long-term epidermal protein expression landscapes after dermal regeneration and in psoriatic inflammation. We first characterized the effects of two dermal regeneration strategies on transplants of indicator split-thickness skin grafts (STSGs) in ten adult patients with deep burns covering more than 20% of their body surface area. After fascial excision, three adjacent areas within the wound were randomized to receive a permanent dermal matrix, a temporary granulation-tissue-inducing dressing or no dermal component as control. Control areas were covered with STSG immediately, and treated areas after two-weeks of dermis formation. Epidermis-dermis-targeted proteomics of one-year-follow-up samples were performed for protein expression profiling. Epidermal expression of axonemal dynein heavy chain 10 (DNAH10) was increased 20-fold in samples having had regenerating dermis vs control. Given the dermal inflammatory component found in our dermal regeneration samples as well as in early psoriatic lesions, we hypothesized that DNAH10 protein expression also would be affected in psoriatic skin samples. We discovered increased DNAH10 expression in inflammatory lesions when compared to unaffected skin. Our results associate DNAH10 expression with cell proliferation and inflammation as well as with the epidermal memory resulting from the previous regenerative signals of dermis. This study (ISRCTN14499986) was funded by the Finnish Ministry of Defense and by government subsidies for medical research.

Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-β)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-β/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy., (Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Cells with slow proliferation kinetics that retain the nuclear label over long time periods-the label-retaining cells (LRCs)-represent multipotent stem cells in a number of adult tissues. Since the identity of liver LRCs (LLRCs) had remained elusive we utilized a genetic approach to reveal LLRCs in normal non-injured livers and characterized their regenerative properties in vivo and in culture. We found that LLRCs were located in biliary vessels and participated in the regeneration of biliary but not hepatocyte injury. In culture experiments the sorted LLRCs displayed an enhanced self-renewal capacity but a unipotent biliary differentiation potential. Transcriptome analysis revealed a unique set of tumorigenesis- and nervous system-related genes upregulated in LLRCs when compared to non-LRC cholangiocytes. We conclude that the LLRCs established during the normal morphogenesis of the liver do not represent a multipotent primitive somatic stem cell population but act as unipotent biliary progenitor cells.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

While the cellular mechanisms of liver regeneration have been thoroughly studied, the role of extracellular matrix (ECM) in liver regeneration is still poorly understood. We utilized a proteomics-based approach to identify the shifts in ECM composition after CCl4 or DDC treatment and studied their effect on the proliferation of liver cells by combining biophysical and cell culture methods. We identified notable alterations in the ECM structural components (eg collagens I, IV, V, fibronectin, elastin) as well as in non-structural proteins (eg olfactomedin-4, thrombospondin-4, armadillo repeat-containing x-linked protein 2 (Armcx2)). Comparable alterations in ECM composition were seen in damaged human livers. The increase in collagen content and decrease in elastic fibers resulted in rearrangement and increased stiffness of damaged liver ECM. Interestingly, the alterations in ECM components were nonhomogenous and differed between periportal and pericentral areas and thus our experiments demonstrated the differential ability of selected ECM components to regulate the proliferation of hepatocytes and biliary cells. We define for the first time the alterations in the ECM composition of livers recovering from damage and present functional evidence for a coordinated ECM remodelling that ensures an efficient restoration of liver tissue.

The lack of primary liver tumor cells has hampered testing of potential chemotherapeutic agents in vitro. To overcome this issue we developed a primary mouse liver tumor cell line K07074. The K07074 cells were immortal, exhibited a biliary phenotype, formed colonies in soft agar and displayed an increase in Hedgehog, Notch and Akt signaling. To study the effect of single and combined inhibition of the liver tumor-related pathways on the growth of K07074 cells we treated these with small-molecule antitumor agents. While the inhibition of Akt and Notch pathways strongly inhibited the growth of K07074 cells the inhibition of Wnt and Hedgehog pathways was less efficient in cell growth suppression. Interestingly, the inhibition of Akt pathway at the level of Akt-Pdpk1 interaction was sufficient to suppress the growth of tumor cells and no significant additive effect could be detected when co-treated with the inhibitors of Wnt, Hedgehog or Notch pathways. Only when suboptimal doses of Akt-Pdpk1 interaction inhibitor NSC156529 were used an additive effect with Notch inhibition was seen. We conclude that the Akt pathway inhibitor NSC156529 is potentially useful as single treatment for liver tumors with hyperactivated Akt signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

During the past decade, the rapid development of new transgenic and knock-in mouse models has propelled epidermal stem-cell research into "fast-forward mode". It has become possible to identify and visualize defined cell populations during normal tissue maintenance, and to follow their progeny during the processes of homeostasis, wound repair, and tumorigenesis. Moreover, these cells can be isolated using specific labels, and characterized in detail using an array of molecular and cell biology approaches. The bacterial enzyme, β-galactosidase (β-gal), the product of the LacZ gene, is one of the most commonly used in vivo cell labels in genetically-engineered mice. The protocol described in this chapter provides a guideline for the isolation of viable murine epidermal cells expressing β-gal, which can then be subjected to further characterization in vivo or in vitro.

The serine/threonine kinase AKT/PKB has a critical role in the regulation of cell proliferation. Because AKT signaling is deregulated in numerous human malignancies, it has become an attractive anticancer drug target. A number of small molecule AKT kinase inhibitors have been developed; however, severe side effects have prevented their use in clinical trials. To find inhibitors of AKT1 signaling with principally novel mechanism of action, we carried out a live cell-based screen for small molecule inhibitors of physical interaction between AKT1 and its primary activator PDPK1. The screen revealed one molecule-NSC156529, which downregulated AKT1 signaling, efficiently decreased the proliferation of human cancer cells in vitro, and substantially inhibited the growth of prostate tumor xenografts in vivo. Interestingly, the treated tumor xenografts exhibited higher expression level of normal prostate differentiation markers but did not show augmented cell death, suggesting that the identified compound primarily enhances the differentiation of malignant cells toward normal prostate epithelium and thus poses as an attractive lead compound for developing novel antitumor agents with less cytotoxic side effects., (©2015 American Association for Cancer Research.)

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

Introduction: Dupuytren's contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches., Methods: We studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR)., Results: We found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types., Conclusions: Based on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis.

Melanocytes possess several functions besides a role in pigment synthesis, but detailed characteristics of the cells are still unclear. We used whole transcriptome sequencing (RNA-Seq) to assess differential gene expression of cultivated normal human melanocytes with respect to keratinocytes, fibroblasts and whole skin. The present results reveal cultivated melanocytes as highly proliferative cells with possible stem cell-like properties. The enhanced readiness to regenerate makes melanocytes the most vulnerable cells in the skin and explains their high risk of developing into malignant melanoma.

Thermally cross-linked glucose-containing electrospun gelatin meshes were studied as possible cell substrate materials. FTIR analysis was used to study the effect of glucose on cross-linking reactions. It was found that the presence of glucose increases the extent of cross-linking of fibrous gelatin scaffolds, which in return determines scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends containing up to 15% glucose. Maximum extent of cross-linking was reached at nearly 20% glucose content. Cross-linking effectively resulted in decreased solubility and increased resistance to enzymatic degradation. Preliminary short-term cell culture experiments indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)