Abstract
Abstract Mesenchymal stem cells (MSCs) are one kind of adult stem cells which can be isolated from diverse sources of tissue, including bone marrow, peripheral blood, umbilical cord, placenta, and adipose tissue. MSCs are multipotent stem cell that can differentiate into various mesodermal cell types, including adipocytes, osteoblasts, chondrocytes, muscle cells, or even ectodermal and endodermal cell types such as neurons and hepatocytes, respectively. Therefore, MSCs have become good potential therapeutic candidates for use in tissue engineering and regenerative medicine. Differentiation of MSCs is usually achieved by using growth factors; however, the procedure is expensive and requires longer periods of time for differentiation. Here, we investigate the effects of manipulating biophysical parameters on MSC differentiation, including alteration of the intracellular cytoskeleton and the extracellular matrix (ECM)/external microenvironment of the stem cell. In the first part, we report on the efficient differentiation of placental-derived multipotent cells (PDMCs)—a type of MSC isolated from human term placenta—into a neural phenotype with use of Y-27632, a clinically compliant small molecular inhibitor of Rho kinase (ROCK) which is a major mediator of the intracellular actin-myosin cytoskeleton. Y-27632 induces a higher percentage of neural-like cells in PDMCs without arresting proliferation or cell cycle dynamics. Y-27632-treated PDMCs express several neural lineage genes at the RNA and protein level, including nestin, MAP2, and GFAP. The effect of the ROCK inhibitor is cell-specific to PDMCs, and is mainly mediated through the ROCK2 isoform and its downstream target, myosin II. In the second part, we investigated the endothelial differentiation capacity of MSCs derived from bone tissue (B-MSC) as mediated by manipulating the extracellular microenviroment in terms of the ECM. We found that fetal pre-osteoblast and adult trabecular bone derived (TB) MSCs cultured in ECM can not only enhance endothelial specific marker expression within 4 hours treatment but also form tubular structures and uptake acetylated low-density lipoproteins, fulfilling the functional criteria for endothelial cells. Moreover, addition of B-MSCs but not other cells significantly enhanced vessel formation in the in vivo chick chorioallantoic membrane assay. Mechanistically, this appears to be due to the upregulation of the endothelial transcription factor forkhead box protein C2 (FOXC2) and its downstream gene v3 integrin/CD61in B-MSCs but not BMMSCs by laminin, a component protein of the ECM. Taken together, our data suggest that manipulation of biophysical parameters of MSCs can influence differentiation capacity, as seen in the rapid induction of a neural or endothelial phenotype with the alteration of cytoskeleton or changing the external culturing environment, respectively. These results provide evidence for using non-biological methods—which are robust and may be more cost-effective—to manipulate MSCs and other stem cells in therapeutic use for tissue engineering and regenerative medicine.