Extracellular matrix | EPFL Graph Search

In biology, the extracellular matrix (ECM), also called intercellular matrix, is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM. The animal extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the ECM. Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest. Each type of connective tissue in animals

An extracellular matrix protein microarray to study endothelial to mesenchymal transformation in mitral valve endothelial cells

Blaise Calpe

In the developing heart, a subset of endothelial cells (ECs) undergo a process of endothelial to mesenchymal transformation (EMT) that leads to the remodeling of the cardiac cushions and the formation of the cardiac valves. Understanding how microenvironmental cues regulate EMT has important implication for the treatment of congenital heart valve diseases and for heart valve tissue engineering. Here a set of extracellular matrix (ECM) proteins and growth factors (GF) were tested to determine the extent of regulation of EMT in adult ovine mitral valve endothelial cells (MVECs). Traditional cell culture dishes present limitations on large scale screening of cells-microenvironments interactions. To address this problem, a high throughput cellular microarray platform was developed. Using a contact printer, 84 different combinations of ECM proteins and GF were microarrayed on a modified glass slide. The slides were seeded with MVECs and immunostained against EMT maker alpha smooth muscle actin ([alpha]-SMA). Fluorescence images of each spot of the microarray were taken with a microscope equipped with an automated stage. These images were analyzed with a custom image processing routine developed with CellProfiler to assess the percentage of cells expressing [alpha]-SMA on each ECM combination.

2011

Temperature impacts human epidermal stem cell behaviour through thermoTRPs and mTOR signalling

Johannes Alexander Mosig

Adult stem cells are characterized by their unique ability to self-renew and give rise to a progeny of specialized/differentiated cells. Therefore, they have a remarkable potential for regenerative medicine. However, the use of adult stem cells is restrained by several obstacles such as limited in vitro amplification and uncontrolled differentiation. Adult stem cells reside in tightly regulated microenvironments termed “niches”. The niche is composed of nursing cells, extracellular matrix and a variety of signalling factors that dynamically regulate stem cell behaviour. Consequently, recapitulating the niche in vitro is fundamental in manipulating adult stem cells. In recent years, the physicochemical microenvironment of the niche (e.g. temperature, pH, oxygen tension) has proven to be as important as classical signalling pathways for the regulation of stem cell fate. Therefore, the aim of this thesis is to understand the influence of temperature variations on human epidermal stem cell behaviour. This effort has been accomplished in three main directions. First, we have contributed to the development of two different telemetric temperature sensors that precisely measure and record temperature in vivo and in vitro. These sensors have allowed us to demonstrate that epidermal keratinocytes are continuously subjected to small physiological temperature variations (3 ± 2oC) in vivo. Then, we have engineered a device that allows a tight and dynamic control of temperature while simultaneously providing real-time imaging of cultured human epidermal stem cells. It is shown that temperature variations of 1oC are not trivial, since they significantly affect human epidermal stem cell growth and proliferation. Second, we have shown that human keratinocytes sense temperature by means of temperature-sensitive calcium channels termed thermoTRPs (Transient Receptor Potential). Consequently, we have monitored intracellular calcium concentrations to evaluate the reaction of epidermal stem cells to thermal or chemical stimulations. The strongest calcium influxes have been observed after temperature drops (from 37 to 32oC) suggesting that human keratinocytes are particularly sensitive to temperature decreases. Furthermore, we have demonstrated the importance of these channels for keratinocyte viability by silencing a thermoTRP channel (i.e. TRPV3) using lentiviral delivery of shRNA. Third, we have demonstrated that thermal signals affecting keratinocytes are integrated through the mTOR (mammalian Target Of Rapamycin) signalling pathway, since cool stimuli (32oC) decreased mTOR complex 1 (mTORC1) kinase activity. Culture at 32oC and 100nm rapamycin treatment affected growth and proliferation of human keratinocyte stem cells similarly, further stressing the inhibitory effects of cool temperatures on mTORC1. We also have observed that inhibition of mTORC1 induced nuclear translocation of mTOR, suggesting a putative transcriptional role for mTOR. To conclude, we have established that prolonged inhibition of mTORC1 favours an apparent maintenance of the stem cell phenotype by reducing clonal conversion, without however stopping stem cell aging. Altogether, our results demonstrate that thermoTRPs are wired to the mTOR signalling in human epidermal stem cells. Furthermore, our work thoroughly supports the hypothesis that temperature is a significant actor of the epidermal stem cell niche as it can directly affect stem cell behaviour.

EPFL2013

Acquisition et recalage informatique d'images confocales de tissus denses

Nicolas Fête

Analysis of the 3D structure of a biological sample is an important step to model cell and tissue dynamics. As a model system, we set up to determine the number of nuclei in a follicular papilla using confocal imaging and 3D computer reconstruction. The follicular papilla is a small mesenchymal ovalshape structure localized at the basis of the hair follicle that is particularly important for hair follicle morphogenesis, and the size of which varies during hair growth. However, little is known on the influence of the hair cycle on the number of cells in a follicular papilla. Fgf5 -/- mice have long hairs (angora phenotype) due to an extended hair cycle, and small dermal papilla. There are several hypotheses to explain the latter observation : there are less cells in Fgf5 -/- dermal papilla than in wild type papilla mutant papilla produces less extra cellular matrix than wild type papilla it is a combination of both To discriminate between these hypotheses, dermal papilla were microdissected from vibrissal follicles occupying the same position on the mystacial pad of same age Fgf5 -/- and wild type mice. Nuclei were stained with a fluorescent dye and the papilla examined under a confocal microscope. Because of dermal papilla thickness, two stacks of images were acquired for each sample (recto and verso acquisitions), registrated using especially implemented computer science program and analyzed. This whole process turned out to be far more complex and cumbersome than anticipated. First, we had to design a special setup in order to be able to make two side scans of samples, second the acquisition time of two stacks of images to cover the entire dermal papilla was rather long, third repositioning and analysis of the images were complex. Nonetheless, the number of nuclei in a dermal papilla was determined with precision. Analysis of dermal papilla obtained at different time of the hair cycle is ongoing. Our work paves the way to the analysis of other biological structures important during skin morphogenesis or regeneration, i.e. the ectodermal placodes. An important lesson of our work is that there is no straightforward imaging and computer science approach when it comes to the biology of complex structures and that the technology needs to be customized to the biological question.

EPFL2008