(A) PEG hydrogels with tunable mechanical properties. Young’s modulus is linearly correlated with precursor polymer concentration; (E; n=4) red circle indicates muscle elasticity. (B) Image of a pliant PEG hydrogel on a green spatula. Scale bar, 7mm (top). Confocal immunofluorescence image of hydrogel microcontact printed with laminin specifically at the bottom of hydrogel microwells (i.e. from the ‘tips’ of the micropillars). Scale bar, 125 μm (bottom). (C) Dissected tibialis anterior muscles (n=5 animals, 10 muscles total) were analyzed by rheometry (horizontal line indicates the mean). (D) Gel surface protein density did not differ significantly on PEG hydrogels of different rigidities (Young’s modulus, E; p>0.05) and was 7.6 ng/cm² ± 1.0 ng/cm² (n≥4). (E) Scheme of Baxter Algorithm analysis of timelapse videos. Hydrogel arrays with hundreds of microwells containing single MuSCs were followed by timelapse microscopy for 3 days. Videos were automatically processed and analyzed (Supplemental Methods). Scale bar, 100 μm. (F) Single MuSC (black data points) velocity on pliant or rigid culture substrates. Circles denote mean velocity ± standard deviation (p<0.0001). (G) Change in total MuSC number on soft (top plot) or stiff (bottom plot) substrates during timelapse acquisition. Deaths (X) and divisions (O) are shown and colors designate five cell generations (G1–G5). The proportion of cells in each generation at all timepoints is shown. Cell number is normalized to a starting population of 100 single MuSCs.

(A) Scheme of in vivo transplantation experiments. (B) Scatter graph of BLI values of recipient mice one month after transplantation with 100 GFP/Fluc MuSCs after 7 day culture on substrates of varying stiffness (left; n=15). Representative bioluminescence images of animals transplanted with each culture condition are shown (right; photons/sec/cm²/sr). (C) Percentage of animals from each experimental condition that had a BLI value above the engraftment threshold. Fisher’s exact test p<0.05. (D) Scatter graph of BLI values of recipient mice one month after transplantation with different numbers of Fluc MuSCs cultured for 7 days on either hydrogel (black) or plastic (red). Representative bioluminescence images of animals transplanted with each culture condition are shown (right; photons/sec/cm²/sr). (E) Percentage of total transplanted animals in each experimental condition exhibiting a BLI value above the engraftment threshold.

(A) Engraftment of freshly isolated (black line, squares; 500 cells) and pliant (green line, circles; 1500 cells) or rigid (red line, diamonds; 1500 cells) substrate cultured MuSCs monitored by BLI for a period of 30 days post transplantation (p<0.05). (B) Immunofluorescence of GFP expression in transverse sections of muscles one month after transplantation with freshly isolated (left; 500 transplanted cells) or one week pliant hydrogel cultured MuSCs (right; 2500 transplanted cells). GFP=green, Laminin=red, and Hoechst=blue. Scale bar, 100μm. (C) Immunohistochemical analysis of transverse muscle tissues one month after transplant with pliant substrate cultured MuSCs. Arrow points to a donor derived cell in satellite cell position. β-galactosidase=green, Laminin=red, and Hoechst=blue. Scale bar, 50μm.

(A) Scheme of in vivo self-renewal assay. (B) Percentage of total doublets exhibiting a Pax7^+/+ gene signature in pliant (n=47) or stiff (n=32) microwells (right). Representative image of a Pax7^+/+ doublet (left). Native Zs-Green=green (i.e. Pax7), Hoechst=blue, scale bar, 20μm. (C) Scatter graph (left) of bioluminescent values from mice transplanted with doublets derived from pliant (n=12) or stiff (n=14) microwells or clones collected from pliant microwells (n=8). Images of animals with bioluminescent values above threshold are shown (right; photons/sec/cm²/sr). (D) Immunohistochemistry of GFP expression in transverse sections of muscles one month after transplantation with five MuSC doublets (top; GFP⁺ fibers persist ~13mm longitudinally) or a single clone (bottom; fibers persist ~7mm longitudinally) cultured on pliant hydrogel. GFP=green, Laminin=red, and Hoechst=blue. Scale bar, μm.