The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates. We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin alpha 5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

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.

The invasion of carcinoma cells is a crucial feature in carcinogenesis. The penetration efficiency not only depends on the cancer cells, but also on the composition of the tumor microenvironment. Our group has developed a 3D invasion assay based on human uterine leiomyoma tissue. Here we tested whether human, porcine, mouse or rat hearts as well as porcine tongue tissues could be similarly used to study carcinoma cell invasion in vitro. Three invasive human oral tongue squamous cell carcinoma (HSC-3, SCC-25 and SCC-15), melanoma (G-361) and ductal breast adenocarcinoma (MDA-MB-231) cell lines, and co-cultures of HSC-3 and carcinoma-associated or normal oral fibroblasts were assayed. Myoma tissue, both native and lyophilized, promoted invasion and growth of the cancer cells. However, the healthy heart or tongue matrices were unable to induce the invasion of any type of cancer cells tested. Moreover, when studied in more detail, small molecular weight fragments derived from heart tissue rinsing media inhibited HSC-3 horizontal migration. Proteome analysis of myoma rinsing media, on the other hand, revealed migration enhancing factors. These results highlight the important role of matrix composition for cancer invasion studies in vitro and further demonstrate the unique properties of human myoma organotypic model. (C) 2016 Elsevier Inc. All rights reserved.

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-beta 1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

Perineuronal nets (PNN) are a special and highly structured type of the CNS extracellular matrix. In past few years, the important role of PNN in the normal physiology of the CNS and the changes in their expression associated with some pathologies have been shown, thus suggesting that PNN are involved in the pathogenesis of a number of brain and spinal cord diseases. Until recently, no quantita-tive studies have focused on the spatial geometry of the PNN meshes. In 2021 and 2022, we published two quantitative studies of the PNN microstructure in the cerebral cortex based on two new, different methods developed by us to analyze high-resolution confocal microscopy data. This article summarizes the results of a comparative analysis of these two methods for quantitative study of the PNN microstruc-ture using microscopy data on the medial prefrontal cortex in an experimental model of schizophrenia. A high correlation was found between the two methods for the mesh area and the linear dimensions of the three-dimensional mesh structure. No correlation was observed for the two-dimensional shape parameters of the mesh. The obtained results demonstrate that the two methods are complementary and have additive value for future quantitative studies of the PNN microstructure.