BackgroundThe growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear.ResultsHere we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures.ConclusionsIn the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0051-y) contains supplementary material, which is available to authorized users.

Cytokeratin 5 (CK5) impacts mammary epithelial cell lineage differentiation CK5 loss at pre-invasive stage causes impaired maturation of myoepithelial cells CK5 loss causes SLUG downregulation and differentiation bias in mammary progenitors

Leptospira infection involves the adhesion of the bacteria followed by invasion of the host crossing the extracellular matrix barrier. In an effort to understand the molecular mechanism of this process, the possibility of occurrence of matrix degrading enzymes from Leptospira was investigated. Zymographic analysis showed that the outer membrane of Leptospires contains a gelatinase of average molecular size of 46 kDa. The gelatinase exhibited maximum activity at neutral pH and was inhibited by metal chelators such as EGTA, EDTA, and Orthophenanthroline and was activated by calcium, magnesium, zinc, and copper, suggesting that it is a membrane-associated neutral matrix metalloproteinase. Analysis of the production of the enzyme by various serovars showed that the pathogenic serovars expressed significant amount of this enzyme while nonpathogenic forms either did not express or showed only very low activity, suggesting that this enzyme may be associated with pathogenesis of leptospirosis.

Leptospirosis is a major public health problem caused by spirochete Leptospira which is an extracellular pathogen. During infection and invasion, the bacteria cross the physical barriers and later it encounter with the host defence mechanism. These processes may involve proteolytic degradation of the host tissue biomatrix. In an effort to understand the production and nature of Leptospiral proteinases, investigations were carried out using zymograpic methods. The results showed that the leptospires degrades different kind of protein substances such as gelatin, casein, and albumin. Gelatin zymography reveals that different serovars contain multiple gelatinases in the molecular weight range from 240 to 32 kDa. Studies using inhibitors suggested that the Leptospiral proteinases include metalloproteinases, serine or cysteine proteinases. The temperature sensitivity suggests that some of these proteinases are stable even at high temperatures. The presence of multiple gelatinases in Leptospira serovars suggests a critical role for these enzymes in Leptospiral invasion and pathogenesis.

Viral vectors enable efficient transfection of ectopic DNA into hard to transfect cells. Viral vectors are normally used to obtain permanent modification of target cells and tissues expect for the cases where integrase deficient viruses are used. Here we describe a method to stably transfect metanephric mesenchyme cells isolated from the murine embryonic kidney at day E11.5. Using this method, it is possible to transfect hard to transfect cells and successfully evade host tissue immune response. Due to these advantages, this method has become one for the most frequently used in generating stable cell line, manipulation of tissues and gene therapy.