Characterization of cell signaling, morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism.

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.

Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

Advances in Liver Cancer Stem Cell Isolation and their Characterization.

Over the last decade research on cancer stem cells (CSC) significantly contributed to a better understanding of tumor biology. Given their similarity to normal stem cells, i.e. self-renewal and pluripotency the need arises to develop robust protocols for the isolation and characterization of CSCs. As with other malignancies, hepatic tumors are composed of a heterogeneous population of cells including liver cancer stem cells (LCSC). Yet, a precise understanding of why stem cells become cancerous is still lacking. There is unmet need to develop robust protocols for the successful isolation of LCSCs from human tissue resection material as to assist in the development of molecular targeted therapies. Here we review the research progress made in the isolation and characterization of LCSCs by considering a wide range of cell surface markers and sorting methods, as applied to side populations, microsphere cultures and the gradient centrifugation method. We emphasize the different fluorescence activated cell sorting methods and the possibility to enrich LCSCs by immunomagnetic beads. We review the specificity of functional assays by considering ABCG transporter and ALDH1 enzyme activities and evaluate the in vivo tumorigenicity of LCSCs in highly sensitive bioassays. Finally, we evaluate different LCSC markers in association with viral and non-viral liver disease and explore the potential of novel drug delivery systems targeting CD133, EpCAM, CD13 and CD90 for the development of molecular targeted therapies. Graphical Abstract.