The dynamic alignment of cells and matrix is critical in many biological processes, including during tissue development and in the progression of a variety of diseases; yet, nearly all in vitro models are static. Thus, it is of great interest to temporally and spatially manipulate cellular alignment to better understand and develop strategies to control these biological processes. Here, strain-responsive buckling patterns on PDMS substrates are used to dynamically and spatially control human mesenchymal stem cell (hMSC) organization. The results indicate that cellular alignment and pattern recognition are strongly diminished with culture time, which can be overcome by limiting cellular proliferation. Preferential alignment of the hMSCs is completely eliminated after the topography switch from patterned to flat, and can be reversibly repeated for at least 8 cycles. The hMSCs are responsive to dynamic changes in pattern size, where the distribution of the cells with preferential alignment increase with increasing pattern amplitude and decreasing wavelength. Furthermore, by introducing a biaxial stretching system, dynamic control is introduced over the cellular orientation angle and order, and by controlling the UV-ozone exposure of the PDMS, the topographical features can be spatially patterned.