Cell morphology plays an important role in controlling cell functions. Application of micropatterned surfaces in cell biology provides reproducible cell morphology and relative stable adhesion and cytoskeleton pattern for investigation of stem cell functions. We have used photo-reactive polymers and UV lithography to prepare micropatterns to control cell size, shape, adhesion area, aspect ratio and chirality to investigate their influences on stem cell differentiation and gene transfection. In this study, the independent influence of adhesion and spreading area on differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using micropatterned surfaces to precisely control cell adhesion and spreading areas.
The micropatterns were prepared by micropatterning non-adhesive PVA on cell adhesive TCPS surface. Ten micropattern structures were designed and prepared to control cell adhesion area and cell spreading area separately. The micropatterns were composed of many TCPS microdots having a diameter of 2 μm in a round circle having a diameter of 70, 60 and 50 μm. The TCPS microdots and round circles were surrounded by PVA. The micropatterns were designed to control the cells to have the same spreading area but different adhesion area, or to have the same adhesion area but different spreading area. MSCs were cultured on the micropatterns. The formation of FAs and the cytoskeletal organization in the cells were investigated to evaluate cell adhesion and spreading state. The mechanical properties of micropatterned cells and the transduction of cytoskeletal force into nucleus were characterized to reveal the mechanism of the influence. The osteogenic and adipogenic differentiation of MSCs on the micropatterned surfaces were evaluated.
When cell spreading area was the same, cells with small adhesion area formed FAs at cell edge. Their cytoskeletal structure was mainly composed of radically assembled DSFs. The lack of myosin binding to DSFs resulted in low cytoskeletal tension. And the YAP/TAZ mainly distributed in cytoplasm. Therefore, cells with small adhesion area preferred to differentiate into adipocytes. Increasing in cell adhesion area reinforced the cell/material adhesion strength. Cells formed integrated actin network including VSFs, DSFs and TAs. Association of myosin with VSFs and TAs generated high cytoskeletal tension. The cytoskeletal tension stimulated accumulation of YAP/TAZ into nucleus. Cells with large adhesion area showed high potential to become osteoblasts. When cell adhesion area was the same, changing spreading area did not significantly affected stem cell fate determination. Cells with the same adhesion area showed similar potential of osteogenic or adipogenic differentiation. The results indicated that the adhesion area rather than spreading area played more important roles in manipulating stem cell functions. Large adhesion area facilitated the osteogenic differentiation, while small adhesion area promoted the adipogenic differentiation.