Fig 1.
Cardiomyocytes were patterned on a cytophobic polyacrylamide surface patterned with covalently bound fibronectin by micro-contact printing. a) Trans-illumination image of patterned cardiomyocytes showing the full 6 mm field of view. b) Fluorescence images of cardiomyocytes on patterned islands with nuclear stain DAPI (blue) and myofibril stain cardiac troponin T (red). c) A magnified view of one 250 μm island. d) Myofibril banding is clearly visible at single-cell resolution.
Fig 2.
Optical electrophysiology recordings.
(a) Fluorescence image of QuasAr2 expressed in 250 μm islands, all of which were monitored simultaneously at a 500 Hz frame rate. b) Fluorescence recordings of QuasAr2, indicating voltage, from islands in (a). The individual islands showed heterogeneity in spontaneous beat rate and action-potential waveform. c) Fluorescence of GCaMP6F expressed in the same islands as in (a). d) Fluorescence recordings of GCaMP6F, indicating Ca2+, from islands in (c). Data in (b) and (d) has been corrected for slow photobleaching, but not otherwise filtered.
Fig 3.
Island size-dependent action potential parameters in hiPSC-CM.
a) Mean spontaneous beat period as a function of island size at 14 dpp. b) Standard deviation of beat period across islands, as a function of island size. c) Amplitude of the calcium transient as a function of island size. d) Amplitude of the voltage transient as a function of island size. e) Rise time (20% to 80%) of the voltage transient as a function of island size. Error bars in a,c-e represent s.e.m.
Fig 4.
RNA sequencing to probe the difference between hiPSC-CM grown in small (100 μm) islands vs. a confluent monolayer.
a) Confluent cells had greater expression of cardiac-related genes, while cells grown on small islands had greater expression of genes related to extracellular matrix, adhesion, and angiogenesis. b) Gene expression differences between small islands and confluent monolayer, projected onto two vectors in gene-expression space. The CP → Adult axis comprises the 3,463 genes that were significantly differentially expressed between cardiac progenitors and adult cardiomyocytes. The Small Islands → Confluent monolayer axis comprises the 149 genes that were significantly differentially expressed between the two island sizes. ESC: embryonic stem cells MES: mesoderm, CP: cardiac progenitors, CM: embryonic stem cell-derived cardiomyocytes.
Fig 5.
Growth on patterned islands identifies factors that influence hiPSC-CM maturation.
a) Cells were cultured on patterned islands with five regions in which different intercellular influences were active. b) Chart showing the modes of intercellular interaction active in each region. c) Image of hiPSC-CM cultured on patterned islands. Scale bar 500 μm. d) Amplitude of calcium transient; e) Amplitude of voltage transient; f) Action potential rise time, for the five regions indicated in (a). In (d-f) measurements were performed on n = 282 samples. Error bars represent s.e.m. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.