Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

Abstract

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Figure 1

Soft substrates promote neuroepithelial conversion while inhibiting neural crest differentiation of hESCs in a BMP4-dependent manner. (a) Schematic diagram showing experimental design of hESC neural induction. hESCs were cultured for 8 d in neural induction medium containing the dual Smad inhibitors, SB 431542 (SB, 10 μM) and LDN 193189 (LDN, 100 nM). (b) Representative immunofluorescence images showing Pax6⁺ NEs and AP2⁺ NCs after 8 d of culture with (top; 100 nM) or without (bottom) LDN on vitronectin-coated coverslips and rigid (E = 1,200 kPa) and soft (E = 5 kPa) PMAs. Scale bar, 100 μm. (c) Bar graph showing percentage of Pax6⁺ NEs on coverslips and rigid and soft PMAs at day 4, 6, and 8. (d) Western blotting showing Pax6 and Sox1 expression levels in hESCs cultured for 8 d on coverslips and rigid and soft PMAs. (e) Bar plot showing percentages of Pax6⁺ NEs and AP2⁺ NCs at day 8 as a function of substrate rigidity and LDN concentration. Data represents the mean ± s.e.m with n = 3. P-values were calculated using one-way ANOVA, followed by Tukey post hoc analysis. *, P < 0.05; **, P < 0.01.

Figure 2

Purity and yield of functional motor neurons (MNs) derived from hESCs are improved on soft substrates. (a) Schematic diagram showing experimental design for sequential neural induction, patterning, and functional maturation of MNs from hESCs. hESCs were cultured on vitronectin-coated coverslips and rigid (E = 1,200 kPa) and soft (E = 5 kPa) PMAs in neural induction medium containing the dual Smad inhibitors SB and LDN for 8 d and then in MN differentiation medium containing purmorphamine (Pur), basic fibroblast growth factor (bFGF), and retinoic acid (RA) for an additional 8 d. Putative MN progenitor cells collected at day 16 were transferred onto coverslips and cultured in MN maturation medium containing brain-derived neurotrophic factor (BDNF), ascorbic acid, cyclic adenosine monophosphate (cAMP), and insulin-like growth factor 1 (IGF-1) for another 14 d. (b) Representative immunofluorescence images showing Tuj1⁺, Isl1⁺, and HB9⁺ cells at day 23 (top), and ChAT⁺ cells at day 30 (bottom). Scale bar, 100 μm. (c&d) Bar plots showing percentages (c) and relative numbers (d) of Tuj1⁺, Isl1⁺, and HB9⁺ cells at day 23 as a function of substrate rigidity. Data in d was normalized to values from coverslips. Data represents the mean ± s.e.m with n ≥3. P-values were calculated using two-side unpaired student t-tests. *: P < 0.05; **: P < 0.01. (e–h) Electrophysiological characterization of functional MNs derived from soft PMAs at day 30 using whole-cell patch clamp. e: spontaneous action potential (AP) with resting membrane potential of −64.5 mV; f: voltage response to current step injection; g: instantaneous frequency change or spike frequency adaptation (SFA) evoked with positive current injection; h: post-inhibitory rebound (PIR) after hyperpolarizing current injection.

Figure 3

Soft substrates promote hESC neuroepithelial conversion through a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling regulated by rigidity-dependent Hippo-YAP activities and the actomyosin cytoskeleton (CSK). (a) Western blotting of total and phosphorylated Smad 1/5/8 (p-Smad 1/5/8) in hESCs cultured in neural induction medium for 3 d on rigid (E = 1,200 kPa) and soft (E = 5 kPa) PMAs. (b&c) Representative immunofluorescence images (b) and bar plot (c) showing rigidity-dependent subcellular localization of YAP in hESCs at day 0 and day 3. Scale bar in b, 50 μm. (d) Western blotting for YAP, TAZ, Smad 1/5/8, and Smad 2/3 in nuclear and cytoplasmic protein fractions from hESCs cultured for 3 d on rigid and soft PMAs. (e) Western blotting for phosphorylated YAP S127 (p-YAP S127) and YAP in whole cell lysates of hESCs after 3 d of culture on rigid and soft PMAs. (f&g) Representative immunofluorescence images (f) and bar plot (g) showing the percentage of Pax6⁺ NEs derived from scramble control and siLats1 knockdown hESCs after 6 d of culture on rigid and soft PMAs. Scale bar in f, 50 μm. (h) Bar graph showing the percentage of hESCs with nuclear localization of YAP after 3 d of culture with different drug supplementations, as indicated. (i&j) Representative immunofluorescence images (i) and bar plot (j) showing the percentage of Pax6⁺ NEs derived from hESCs on rigid and soft PMAs after 6 d of culture with different drug supplementations, as indicated. Scale bar in i, 50 μm. In Western blots β-actin and GAPDH were used as protein loading controls and lamin A/C for nuclear fraction control. Data represents the mean ± s.e.m with n ≥ 3. P-values in c & g and h & j were calculated using two-side unpaired student t-tests and one way ANOVA followed by Tukey post hoc analysis, respectively. *, P < 0.05; **, P < 0.01.