Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells

Abstract

Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness “pulse,” which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.

Biomaterials for Enhancing Neuronal Repair

“…Such measurements of human brain tissue are highly dependent on frequency and region, and therefore variable. Additional studies have demonstrated that timing and duration of exposure to stiffness cues impacts stem cell differentiation to neural cell types, and that while neuritogenesis may be enhanced on soft substrates, network connectivity and signal transduction are enhanced by stiffer substrates (Balgude et al, 2001;Jiang et al, 2010;Keung et al, 2012;Zhang et al, 2014;Mosley et al, 2017). These findings emphasize that stiffness cues should be adjusted depending on the desired outcome, with close attention to the region of interest in the human body.…”

“…b Electrical References: (Jaffe and Stern, 1979;Patel and Poo, 1982;Hotary and Robinson, 1991;Davenport and McCaig, 1993;Metcalf and Borgens, 1994;Yao et al, 2008Yao et al, , 2009Yao et al, , 2011Graves et al, 2011;Koppes et al, 2014;Kim et al, 2016;Ma et al, 2016). c Mechanical Stiffness References: (Balgude et al, 2001;Discher et al, 2005;Jiang et al, 2010;Keung et al, 2012;Lee et al, 2013;Zhang et al, 2014;Mosley et al, 2017).…”

Tissue mechanics regulate brain development, homeostasis and disease

“…1). As might be expected given the importance of the mechanical properties of the in vivo niche (discussed above), pluripotent embryonic stem cells (ESCs) favor neurogenesis when plated on substrates that resemble soft brain tissue (Keung et al, 2012), and mesenchymal stem cells (MSCs) also upregulate neuronal markers when cultured on soft substrates in the Young's modulus range of 0.1-1.0 kPa (Engler et al, 2006). Studies testing the effect of niche mechanics on the behavior of NSCs isolated directly from the SVZ show that NSCs on softer substrates that mimic neurogenic brain regions, such as the dentate gyrus of the hippocampus, tend to differentiate into neurons, whereas cells on substrates of increased stiffness foster glial differentiation (Saha et al, 2008;Georges et al, 2006;Leipzig and Shoichet, 2009).…”

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain

“…Its stiffness in the postnatal stage has been examined previously in the rodent cerebral cortex (Elkin et al, 2010), the hippocampus (Elkin et al, 2007) and the cerebellum (Christ et al, 2010), although it has never been measured at developing stages (Franze, 2013). Interestingly, previous studies have demonstrated that culturing mesenchymal stem cells (Engler et al, 2006) and human pluripotent stem cells (Keung et al, 2012) on material that mimics the softness of the brain results in the induction of neural fate. Moreover, lineage switching from neural to glial cells can be influenced by the shift of substrate stiffness (Saha et al, 2008;Leipzig and Shoichet, 2009).…”