Initial Priming on Soft Substrates Enhances Subsequent Topography-Induced Neuronal Differentiation in ESCs but Not in MSCs

Differentiation of stem cells into neurogenic lineage is of great interest for treatment of neurodegenerative diseases. While the role of chemical cues in regulating stem cell fate is well appreciated, the identification of physical cues has revolutionized the field of tissue engineering leading to development of scaffolds encoding one or more physical cues for inducing stem cell differentiation. In this study, using human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs), we have tested if stiffness and topography can be collectively tuned for inducing neuronal differentiation by culturing these cells on polyacrylamide hydrogels of varying stiffness (5, 10, and 20 kPa) containing rectangular grooves (10, 15, and 25 μm in width). While hMSCs maximally elongate and express neuronal markers on soft 5 kPa gels containing 10/15 μm grooves, single mESCs are unable to sense topographical features when cultured directly on grooved gels. However, this inability to sense topography is rescued by priming mESCs initially on soft 1 kPa flat gels and then replating these cells onto the grooved gels. Compared to direct culture on the grooved gels, this sequential adaptation increases both viability as well as neuronal differentiation. However, this two-step process does not enhance neuronal marker expression in hMSCs. In addition to highlighting important differences between hMSCs and mESCs in their responsiveness to physical cues, our study suggests that conditioning on soft substrates is essential for inducing topography-mediated neuronal differentiation in mESCs