Spatially patterned matrix elasticity directs stem cell fate

Abstract

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

Keywords: human mesenchymal stem cell; photodegradable hydrogel; spatial matrix stiffness.

Fig. 1.

(A) Chemical structures of the photodegradable cross-linker (PEGdiPDA) and PEGA. Acrylate functional groups are labeled in red and the photodegradable nitrobenyzl ether is labeled in blue. (B) Photodegradable hydrogels polymerized from PEGdiPDA and PEGA monomers can be softened from stiff (E = 9.6 ± 0.2 kPa) to soft (E = 2.3 ± 0.2 kPa) moduli by irradiation of 365 nm light for 360 s. (C) Immunostaining of hMSCs on stiff and soft hydrogel surfaces. On stiff hydrogels, hMSCs expressed tensile F-actin bundles and had 90.9 ± 4.4% nuclear YAP activation; whereas, on soft hydrogels, hMSCs only had 6.6 ± 3.5% nuclear YAP activation with less organized F-actin structure. DAPI (4’,6-diamidino-2-phenylindole; blue) F-actin (red), YAP (green). Scale bars = 20 μm; n = 5 with over 100 cells analyzed for each condition.

Fig. 2.

(A) An illustration of hMSCs seeded on mechanically patterned hydrogel surfaces with different stiff-to-soft ratios. Black indicates chrome-covered areas that will remain stiff, and white squares indicate areas exposed to light that will be degraded to soft regions. (B) AFM elastic moduli maps of (i) 75% stiff (regular pattern), (ii) 11% stiff (regular pattern), and (iii) 75% stiff (random pattern) hydrogels. (iv) Representative force-deformation data and model fits for two regions in iii. Scale bars = 2 μm.

Fig. S1.

hMSCs spreading area on 2-kPa hydrogels. The average area of a spread hMSC is about 1,500 μm² on uniformly soft substrates; n > 5 with over 100 cells analyzed in each condition. Mean ± SE.

Fig. S2.

AFM elastic moduli maps of uniformly stiff and soft hydrogels. (A) Elastic modulus of the uniformly stiff hydrogel is 12.4 ± 0.2 kPa. (B) Elastic modulus of the uniformly soft hydrogel is 2.0 ± 0.2 kPa. Scale bar = 2 μm.

Fig. S3.

Surface roughness measured by AFM. Images are color coded by surface topographic features, with darker colors representing lower z positions and brighter color represents higher z positions. (A) Root mean square roughness of the uniformly stiff hydrogel is 12 ± 1 nm. (B) Root mean square roughness of the uniformly soft hydrogel is 23 ± 2 nm. (C) Root mean square roughness of the patterned hydrogel (75% stiff) is 43 ± 2 nm. Scale bar = 1 μm.

Fig. 3.

(A, i) Paxillin staining (green) of hMSCs on stiff hydrogels illustrates clustered structures, which indicate the formation of mature focal adhesion. (ii) However, paxillin staining of hMSCs seeded on soft hydrogels results in a basal uniform expression throughout the cell body. Paxillin (green); DAPI (blue). Scale bars = 20 μm. (B) Relative localization of focal adhesions in hMSCs to mechanical regions on the patterned hydrogel. (i) Immunostaining of hMSCs on a regularly patterned hydrogel with 11% stiff area. Focal adhesion formation was observed by staining for paxillin (green), and cytoskeletal organization was observed by staining for F-actin (red). The localization of focal adhesions relative to the mechanically patterned regions was analyzed to identify the relative paxillin intensity within stiff, soft, and interfacial regions based on the fluorescent and DIC channels (Right). For quantification, all paxillin intensities for each image were normalized to the absolute value found in the soft regions. (ii) Immunostaining of hMSCs on regularly patterned hydrogel with 75% stiff area. (iii) Immunostaining of hMSCs on randomized patterned hydrogel with 75% stiff area. In all three conditions, paxillin intensities were about threefold higher in the stiff and interfacial regions relative to the soft regions. Paxillin (green), F-actin (red), DAPI (blue). Scale bars = 20 μm; n > 10 with over 50 cells analyzed for each condition. Data plotted as mean ±SE.

Fig. S4.

Outlining stiff, soft, and interfacial edge regions by image analysis. (A) A representative bright field image of a regularly patterned 75% stiff hydrogel. Image analysis was performed on A to generate B, which was segmented into three regions: interface (white), soft (black inside the interface), and stiff (black outside of the interface) areas. (C) A representative bright field image of a randomly patterned 75% stiff hydrogel. Image analysis was performed on C to generate D, which was segmented into three regions: interface (white), soft (black inside the interface), and stiff (black outside of the interface) areas. Scale bar = 20 μm.

Fig. S5.

Design of the random patterns of 11%, 25%, 50%, and 75% stiff regions. The stiffness of each 2 μm by 2 μm regions was randomly decided based on a random number generator (1 as stiff and 0 as soft) with a defined stiff to soft ratio (11–75% stiff) in the 50 μm by 50 μm (2,500 μm²) square.

Fig. 4.

(A, i) Intracellular YAP (green) localization within hMSCs on regularly patterned hydrogels with 11% stiff area. Image was overlaid with the DIC channel to indicate the underlying pattern. YAP was mainly deactivated in the cytoplasm on 11% stiff hydrogels. (ii) Intracellular YAP localization of hMSCs on regularly patterned hydrogels with 75% stiff area. YAP was observed to primarily be activated in the nuclei. (iii) Intracellular YAP localization of hMSCs on randomly patterned hydrogels with 75% stiff area. YAP was found to be mainly deactivated in the cytoplasm. YAP (green), DAPI (blue). Scale bars = 20 μm. (B, i) The percent of hMSCs with intracellular YAP activation was quantified based on immunostaining analysis and found to increase correspondingly to stiff percentages on regular stiff patterns, indicated by the blue dash line and symbols. A sigmoidal response was observed with a significant increase in YAP activation from 25% stiff to 50% stiff. However, on randomly patterned hydrogels intracellular YAP activation in hMSCs was insensitive to the change of underlying stiff percentages, indicated by the red dash line and symbols; n > 5 with over 100 cells analyzed for each condition; * compared with regular 25% stiff, P < 0.01 based on one-way ANOVA analysis using Turkey’s multiple comparisons test; # compared with random 75% stiff, P < 0.001 based on unpaired t test. (ii) Comparison of relative mRNA expression levels of the genes CTGF and ANKRD1 for hMSCs on regularly and randomly patterned hydrogels with 75% stiff regions. For both genes, hMSCs on regular patterns had significantly higher expression levels than those on the random patterns. n = 3. # P < 0.05, based on unpaired t test.

Fig. 5.

(A) Cell spreading area increased correspondingly to stiff percentages on regularly patterned hydrogels, indicated by the blue dashed line and symbols, consistent with the trend of intracellular YAP activation. A significant increase of cell area was observed from 50% to 75% stiff gels. On the other hand, cell spreading was insensitive to the change of underlying stiff percentages on randomly patterned hydrogels, indicated by the red dashed line and symbols; * compared with regular 50% stiff, P < 0.01 based on one-way ANOVA using Tukey’s multiple comparisons test. (B) Complimentary, cellular circularity decreased relative to stiff percentages on regularly patterned hydrogels, indicated by the blue dashed line and symbols. However, circularity did not change significantly with increased stiff area on randomly patterned hydrogels, indicated by the red dash line and symbols; # compared with 75% regular stiff, P < 0.01 based on one-way ANOVA; n > 5 with over 100 cells analyzed for each condition. Mean ± SE.

Fig. 6.

(A) ALP staining of hMSCs on uniformly stiff, 75% stiff with regular and random patterns and uniformly soft hydrogels after 7 d culture in mixed media. hMSCs on uniformly stiff and regularly patterned 75% stiff samples had prominent ALP expression; but the expression levels were significantly lower on the randomly patterned 75% stiff and uniformly soft samples. ALP (purple). Scale bar = 100 μm. (B) CD105 staining of hMSCs on uniformly stiff, 75% stiff with regular and random patterns and uniformly soft hydrogels after 7 d culture in mixed media. hMSCs on uniformly soft and randomly patterned 75% stiff samples had significantly higher CD105 expressions than those on uniformly stiff and regularly patterned 75% stiff samples. CD105 (green), DAPI (blue). n = 3. Scale bar = 20 μm.

Fig. S6.

Characterization of RGD concentration after irradiation. (A) quantification of the percent release of methacrylated-rhodamine from PEGdiPDA after 360, 600, and 900 s of irradiation. **P < 0.01; ***P < 0.001. (B) Representative images of YAP activation on both 10 and 2 kPa hydrogel with 1.25 mM RGD peptide. (C) Cell spread area on 2 kPa hydrogel with 1.25 and 2.5 mM RGD. (D) Circularity of cell shape on 2 kPa hydrogel with 1.25 and 2.5 mM RGD. N.S. = not significant based on unpaired t test.

Fig. S7.

YAP activation in hMSCs on fibronectin patterned surface. Fibronectin was patterned onto regular 89%, 25%, and randomized 25% surface area in nondegradable PEG-diacrylate hydrogel. YAP was observed to be activated in hMSCs independently of the percentage or organization of the patterned adhesive area. Scale bar = 20 μm.

Fig. S8.

YAP activation and cell morphology of hMSCs on regular 75% stiff substrate treated with Y-27632 ROCK inhibitor. (A) Nuclear YAP activation in hMSCs was significantly reduced from 83.4 ± 6.9% to 12.7 ± 5.4% upon ROCK inhibition. (B) Representative images of YAP localization and F-actin structure in hMSCs on regular 75% stiff substrate treated with Y-27632 ROCK inhibitor. Scale bar = 20 μm. (C) Cell area reduced from 4429 ± 757 to 2173 ± 235 μm² upon ROCK inhibition. (D) Circularity increased from 0.096 ± 0.013–0.2674 ± 0.007 upon ROCK inhibition.