Mechanical memory and dosing influence stem cell fate

Abstract

We investigated whether stem cells remember past physical signals and whether these can be exploited to dose cells mechanically. We found that the activation of the Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) as well as the pre-osteogenic transcription factor RUNX2 in human mesenchymal stem cells (hMSCs) cultured on soft poly(ethylene glycol) (PEG) hydrogels (Young's modulus E ~ 2 kPa) depended on previous culture time on stiff tissue culture polystyrene (TCPS; E ~ 3 GPa). In addition, mechanical dosing of hMSCs cultured on initially stiff (E ~ 10 kPa) and then soft (E ~ 2 kPa) phototunable PEG hydrogels resulted in either reversible or--above a threshold mechanical dose--irreversible activation of YAP/TAZ and RUNX2. We also found that increased mechanical dosing on supraphysiologically stiff TCPS biases hMSCs towards osteogenic differentiation. We conclude that stem cells possess mechanical memory--with YAP/TAZ acting as an intracellular mechanical rheostat--that stores information from past physical environments and influences the cells' fate.

Figure 1. Mechanical dosing and memory of hMSCs

a, hMSCS were cultured on TCPS (blue) in growth media (red) for 1 to 7 days prior to collection and analysis (green, day 0). b, RUNX2 gene expression in hMSCs with mechanical dosing on TCPS as quantified by qRT-PCR. c, hMSCs were cultured on TCPS (blue) in growth media (red) for 1 to 7 days prior to trypsinization and transfer (light green, day 0) to soft hydrogels (orange). hMSCs were cultured subsequently on soft hydrogels in growth media for 3 days prior to collection and analysis (green, day 3). d, YAP and RUNX2 nuclear localization in hMSCs after 3 days on soft hydrogels with previous mechanical dosing on TCPS (DT1 / So3 to DT7 / So3). e, YAP localization in hMSCs on soft hydrogels with 1 day of mechanical dosing on TCPS (DT1 / So3) and 7 days of mechanical dosing on TCPS (DT7 / So3). DAPI, blue; YAP, green. Scale bars, 20 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to DT1 or DT1 / So3.

Figure 2. Influence of phototunable substrate modulus on transcription factor activation

a, Photodegradable hydrogels were fabricated from the free radical polymerization of a photodegrable crosslinker, PEGdiPDA, with a monoacrylated PEG, PEGA. Polymerization results in a stiff hydrogel (~ 10 kPa) that activates YAP in hMSCs (green nucleus, blue cytoplasm). Light exposure (λ = 365 nm; I₀ = 10 mW/cm²) for 360s in the presence of cells softens the substrate to a soft hydrogel (~ 2 kPa), upon which YAP de-activates in hMSCs (blue nucleus, green cytoplasm). b, Light exposure can be used to fabricate culture substrata with a range of moduli by exposing the samples to defined doses of light. In this work, hydrogels with average moduli of ~ 10, 6, 4, and 2 kPa were generated. c, Comparison of Young’s moduli of TCPS, stiff hydrogel, and soft hydrogel. d, YAP activation in hMSCs (nuclear localization) increased with increasing modulus. e, Similarly, RUNX2 activation in hMSCs (nuclear localization) increased with increasing modulus. f, YAP and RUNX2 were both excluded from the nucleus (de-activated) in hMSCs on soft hydrogels (2 kPa). g, YAP and RUNX2 were both localized to the nucleus (activated) in hMSCs on stiff hydrogels (10 kPa). DAPI, blue; YAP, green; RUNX2, red. Scale bars, 20 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3. Reverisble and irreversible effects of mechanical dosing on phototunable hydrogels

a, hMSCs were cultured on stiff hydrogels (orange crosshatch) in growth media (red) for 1 to 10 days prior to in situ softening (purple, day 0) the underlying culture substrata to soft hydrogels (orange). hMSCS were cultured subsequently on the softened hydrogels for 1 to 10 days in growth media prior to collection and analysis (green). b, YAP and RUNX2 response to in situ softening after 1 day of mechanical dosing on stiff hydrogels. Stiff control is the average hMSC expression of YAP or RUNX2 over 3, 5, 7, and 10 days on stiff hydrogels and demonstrates full activation. Soft control is the average hMSC expression of YAP or RUNX2 over 3, 5, 7, and 10 days of soft hydrogels and demonstrates basal levels of activation (Supplementary Fig. S2). After softening, YAP and RUNX2 demonstrated transient activation (DSt1-So1) but de-activated to basal levels by day 3 (DSt1-So3). De-activation persisted to day 5 (DSt1-So5). With 1 day of mechanical dosing on stiff hydrogels, hMSCs demonstrated a transient and fully reversible activation of YAP and RUNX2. c, YAP and RUNX2 response to in situ softening after 7 days of mechanical dosing on stiff hydrogels. Upon softening, YAP remained above basal levels out to 5 days after softening (DSt7-So5). On the other hand, RUNX2 activation relaxed to basal levels by day 5 after softening (DSt7-So5). With 7 days of mechanical dosing on stiff hydrogels, hMSCs demonstrated a partially reversible activation of YAP and RUNX2. d, YAP and RUNX2 response to in situ softening after 10 days of mechanical dosing on stiff hydrogels. 10 days after softening, YAP and RUNX2 persisted at active levels significantly above basal levels for soft hydrogels (DSt10-So1 to DSt10-So10). Thus, 10 days of mechanical dosing on stiff hydrogels induced an irreversible activation of YAP and RUNX2. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4. Influence of mechanical dosing on differentiation of hMSCs

a, hMSCs were cultured on TCPS (blue) in growth media (red) for 1 to 10 days prior to trypsinization and transfer (light green, day 0) to soft hydrogels (orange). hMSCs were cultured subsequently on soft hydrogels for 7 days in mixed media (dark purple) prior to collection and analysis (green, day 7). Control samples were cultured on soft hydrogel in mixed media without mechanical dosing on TCPS (So7). b, PPARγ gene expression in hMSCs with mechanical dosing on TCPS prior to culture on soft hydrogels as quantified by qRT-PCR. c, ALP gene expression in hMSCs with mechanical dosing on TCPS prior to culture on soft hydrogels as quantified by qRT-PCR. d, Immunocytochemistry of PPARγ (green) and osteocalcin (OCN; red) in hMSCs after 7 days on soft hydrogels with various mechanical dose on TCPS. e, Staining for alkaline phosphatase (ALP) in hMSCs with mechanical dosing on TCPS prior to culture on soft hydrogels. f, Representative image of staining for Oil Red O in hMSCs on soft hydrogels. g, Quantification of the percentage of ALP positive cells as a function of mechanical dose on TCPS prior to culture on soft hydrogels. Scale bars, 20 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to So7.