Substrate elasticity induces quiescence and promotes neurogenesis of primary neural stem cells—A biophysical in vitro model of the physiological cerebral milieu

Abstract

In the brain, neural stem cells (NSC) are tightly regulated by external signals and biophysical cues mediated by the local microenvironment or “niche.” In particular, the influence of tissue elasticity, known to fundamentally affect the function of various cell types in the body, on NSC remains poorly understood. We, accordingly, aimed to characterize the effects of elastic substrates on critical NSC functions. Primary rat NSC were grown as monolayers on polydimethylsiloxane‐ (PDMS‐) based gels. PDMS‐coated cell culture plates, simulating the physiological microenvironment of the living brain, were generated in various degrees of elasticity, ranging from 1 to 50 kPa; additionally, results were compared with regular glass plates as usually used in cell culture work. Survival of NSC on the PDMS‐based substrates was unimpaired. The proliferation rate on 1 kPa PDMS decreased by 45% compared with stiffer PMDS substrates of 50 kPa (p < 0.05) whereas expression of cyclin‐dependent kinase inhibitor 1B/p27Kip1 increased more than two fold (p < 0.01), suggesting NSC quiescence. NSC differentiation was accelerated on softer substrates and favored the generation of neurons (42% neurons on 1 kPa PDMS vs. 25% on 50 kPa PDMS; p < 0.05). Neurons generated on 1 kPa PDMS showed 29% longer neurites compared with those on stiffer PDMS substrates (p < 0.05), suggesting optimized neuronal maturation and an accelerated generation of neuronal networks. Data show that primary NSC are significantly affected by the mechanical properties of their microenvironment. Culturing NSC on a substrate of brain‐like elasticity keeps them in their physiological, quiescent state and increases their neurogenic potential.

Substrate Elasticity Exerts Functional Effects on Primary Microglia

“…We previously proposed polydimethylsiloxane (PDMS)based substrates as a suitable in vitro model for the investigation of mechanical influences, given smooth surface topography and excellent biocompatibility (Schellenberg et al, 2014;Abraham et al, 2019;Blaschke et al, 2019). We here hypothesize that the ubiquitous influence of substrate elasticity modulates primary microglia functions and might even alter dynamic microglia reaction upon subsequent stimuli.…”

“…We and others have previously shown that a simulation of mechanical properties on CNS cells in vitro is feasible and allows analyzing cell functions under more physiological conditions than provided by regular cell cultures, uncovering essential aspects and mechanobiological properties of neural stem cells and neurons (Pathak et al, 2014;Abraham et al, 2019;Blaschke et al, 2019). Overall, mechanical properties alter the development and behavior not only of mesenchymal stem cells (Murphy et al, 2011), hematopoietic stem cells (Kumar et al, 2013), and cardiomyocytes (Hersch et al, 2013) but also of neural cells like neurons (Abraham et al, 2019), astrocytes (Moshayedi et al, 2014), and neural stem cells (Blaschke et al, 2019). While single reports describe morphological alterations of microglia dependent on underlying substrate elasticity (Moshayedi et al, 2014;Bollmann et al, 2015), the impact of elasticity on microglia at the functional level to date remains elusive.…”

Physical Cues of Matrices Reeducate Nerve Cells

“…The stiffness of the matrix mainly affects the differentiation of NSCs into different cell phenotypes. When the stiffness of the matrix is approximately 1 kPa, NSCs are more easily differentiated into neurons ( Rammensee et al, 2017 ; Blaschke et al, 2019 ). Leipzig and Shoichet (2009) developed a photopolymerized methylacrylamide chitosan biomaterial with a Young’s modulus adjustable from less than 1 kPa to greater than 30 kPa.…”

“…On 1 kPa PDMS, the number of neurons was 42%, while on 50 kPa PDMS, the number of neurons was 25%. Neurons from NSCs on 1 kPa PDMS showed 29% longer neurites compared with those on stiffer PDMS substrates, suggesting optimized neuronal maturation and an accelerated generation of neuronal networks ( Blaschke et al, 2019 ). If the stiffness of the matrix exceeds or decreases below a certain range (∼1 kPa), the proportion of neurons will decrease.…”

“…Furthermore, the response of NSCs to the stiffness of the matrix is not particularly sensitive and requires a larger span to be apparent. When the stiffness of the matrix was 15 and 50 kPa, the proportion of neurons was equal ( Saha et al, 2008 ; Blaschke et al, 2019 ). In terms of NSC differentiation into glial cells, in general, the differentiation ability increased as the stiffness of the matrix increased.…”