Figure 5
From: Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns
Assessing the role of CFBs on the alignment of iPS-CMs as a function of the fibronectin micropattern. (A) Representative images of iPS-CMs with 10% CFBs on the fibronectin micropatterns. CFBs can be distinguished from cardiomyocytes by the lack of α-actinin (red) staining. (B) Representative images of iPS-CMs with 33% CFBs on the fibronectin micropatterns. Note the increase in cell alignment relative to (A). (C) CFBs on the fibronectin micropatterns at low density show pattern-dependent alignment. (D) CFBs on the fibronectin micropatterns at high density show alignment on all patterns. (E) Fluorescent staining of fibronectin generated by CFBs cultured on the micropatterned substrates, after CFB removal. The 20 × 20 pattern has significantly less CFB-generated fibronectin, likely due to the weaker attachment to the original fibronectin pattern. (F) Representative images iPS-CMs cultured on the CFB-generated ECM shown in (E). (G) Alignment analysis of iPS-CMs on the fibronectin micropatterns with 0%, 10% or 33% addition of CFBs shows increased OOP with increased CFB percentage for all patterns. (H) Alignment analysis of CFBs on the fibronectin micropatterns shows that at low density CFB OOP is pattern-dependent, but at high density it is comparably high for all three patterns. (I) Alignment analysis of fibronectin generated by CFBs after CFB removal from the surface (CFB-ECM) reveals that for the 2 × 2 and biomimetic patterns, OOP for CFB-ECM is much lower than for the CFB actin cytoskeleton. (J) Alignment analysis of iPS-CMs cultured on the CFB-ECM shows decreased OOP for the 2 × 2 and biomimetic conditions compared to the iPS-CM controls cultured on the fibronectin micropatterns alone. Scale bars are 50 µm. (H), (J) Two-way ANOVA with Tukey post hoc, *indicates p < 0.05, n ≥ 3. (G), (I) One-way ANOVA with Tukey post hoc, *indicates p < 0.05, n ≥ 3
