The effect of mesenchymal stem cell shape on the maintenance of multipotency.

Human mesenchymal stem cells (MSCs) have broad therapeutic potential due to their ability to differentiate into multiple cell types. However, when cultured ex vivo MSCs will spontaneously differentiate and have been shown to lose multipotency after prolonged passaging. Cell culture conditions that promote maintenance of multipotency during in vitro expansion are a critical need to fully realize the therapeutic potential of MSCs. Here we show that by confining MSCs to small islands, we can restrict inappropriate lineage specification and enhance the expression of mesenchymal stem cell markers Stro-1 and Endoglin. Even when released from the islands and reseeded, cells previously cultured in patterns maintain higher expression of MSC markers compared to cells cultured on plastic, while maintaining their ability to differentiate into adipocytes and osteoblasts. Exposure of non-patterned cells to inhibitors of myosin and Rho-associated protein kinase (ROCK) leads to increased expression of stem cell markers. Our findings suggest that maintenance of MSC "stemness" requires a low state of actomyosin contractility. This work will prove useful in the development of culture conditions for the maintenance of multipotent MSCs in vitro and for the design of niche-mimetic biomaterials.