Characterization of the Frustrated Differentiation of Mesenchymal Stem Cells Induced by Normadic Migration Between Stiff and Soft Region of Gel Matrix

Abstract

Mesenchymal stem cells (MSCs) have been known to exhibit substrate stiffness-dependent differentiation, and history of the mechanical dose from culture environment to MSCs sensitively is found to alter its phenotype. A certain level of substrate stiffness and duration period on that determine the fate of MSCs. In relation to this, we have found before that microelastically-patterned hydrogel with heterogeneous distribution of matrix stiffness allow MSCs to suppress fate determination into specific differentiation lineages, and contribute to keep the undifferentiated state. We call such mode of MSCs as “frustrated differentiation”, which serves to construct culture substrate for MSCs to maintain their stemness in high-qualified state. The basis of this phenomenon is in the enforced oscillation of mechanical dose from environment to MSCs during the nomadic migration between stiff and soft region of gel matrix, which eliminate the history of experience on a certain level of stiffness. To design such heterogeneous microelastically-patterned gels, we have applied the photolithographic microelasticity patterning using photoculable gelatins. The emergence of frustrated mode of differentiation was previously confirmed by immunofluorescence and RT-PCR analysis for the expression markers, but more detailed and precise characterizations are of course required. In this study, to fully characterize the frustrated differentiation of MSCs, we investigated oscillation of the mechanical dose and mechanical signal input to MSCs employing the long-term traction force microscopy for MSCs culture on the microelastically-striped patterned gels. In addition, we performed cDNA microarray analysis for the MSCs culture in such mode of frustrated differentiation. As the result, MSCs in normadic movement between stiff and soft region of gel surface were confirmed to exhibit characteristic transcriptome and marked large fluctuation profile of traction forces compared with plain control gels.