Figure 2. Formation of cardiac fibers from mESC-CM and WT19 fibroblasts.

(a) A 50:50 mixture of mESC-CM cells (green) and WT19 fibroblasts (red) seeded in a Matrigel pillow led to the formation of regularly contracting engineered cardiac fibers starting on day 3. Cells were preloaded with live cell indicators before seeding. The fibers continued to contract for >10 d after plating. The right panel shows a robust motion artifact associated with repetitive contractions of the fibers throughout acquisition period of a high resolution confocal image. Identical conclusions were derived from n = 4 independent mESC-CM and WT19 co-culture experiments.

(b) Phase contrast image showing the typical appearance of spontaneously beating engineered cardiac fibers made of 1:1 cell mixture of fibroblast and cardiac myocytes. The right panel shows representative traces from one Fluo-4 loaded fiber. Signals were acquired from three different regions of interest, placed at ~200 μm from each other. Each transient corresponds to a single contraction. Synchrony between the individual traces indicates that cardiac myocytes within the construct are electrically well-coupled to each other. When seeded in a Matrigel pillow in the absence of fibroblasts, mESC-CM failed to form 3D cardiac fibers. Instead, mESC-CM formed confluent, flat monolayers indistinguishable in appearance from cells plated on laminin-coated coverslips.