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

Abstract

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.

The effects of surface topography modification on hydrogel properties

“…With the priming step, neuronal differentiation was improved in mESC, and the authors suggested that soft substrates are essential for inducing topography-mediated neuronal differentiation in mESCs. 204 The observations were in agreement with earlier studies to show that cytoskeletal contractility is essential for topography-sensing and topography-induced neuronal differentiation of human ESCs. 205 Undifferentiated pluripotent stem cells including hESCs 205 and mESCs 206 have lower acto-myosin contractility compared to differentiated cells, while the acto-myosin contractility increased during differentiation process.…”

Recent Developments in Surface Topography-Modulated Neurogenesis

“…Since the piezoelectric material generated an electric potential by converting mechanical forces such as cell movement and traction, rat bone marrow-derived MSCs were directly seeded onto the substrates without further electrical stimulation. Based on the data collected from the study, when 200 [55] published by American Chemical Society 2019, reproduced with permission from [56] published by Elsevier 2020) ◂ and 500 nm patterned polyvinyl chloride (PVC) was used as non-electrical control, nanotopography was sufficient to promote the cellular adhesion of the stem cells. However, only the combination with PVDF could greatly enhance the neuron-like differentiation of the MSCs at the 200 nm pattern.…”

The Effect of Physical Cues of Biomaterial Scaffolds on Stem Cell Behavior

“…However, after priming mESCs on a softer flat gel (1 kPa), their viability and neural differentiation were enhanced, whereas hMSCs did not respond to such priming. [97] To mimic the in vivo physical properties and/or to understand more delicate details of stem cell differentiation, recent studies have been investigating how the change/gradient of stiffness can affect stem cell differentiation and migration. Limitations exist, however, in producing hydrogels with stiffness gradients, such as cost, complex techniques, small stiffness ranges, poor biocompatibility, and lack of reproducibility.…”

“…studied the collective effects of substrate stiffness and topography on neural differentiation of hMSCs and mESCs. [ 97 ] Unlike hMSCs that expressed neural markers on 5 kPa gels with grating topography, mESCs did not differentiate. However, after priming mESCs on a softer flat gel (1 kPa), their viability and neural differentiation were enhanced, whereas hMSCs did not respond to such priming.…”

“…However, after priming mESCs on a softer flat gel (1 kPa), their viability and neural differentiation were enhanced, whereas hMSCs did not respond to such priming. [ 97 ]…”