Figure 2.
Workflow of EMLOC induction, polarization, and maturation stages
(A) H3.1.1 low passage hiPSCs broken out on Matrigel after cryopreservation.
(B) Expanded hiPSC colony in mTeSR Plus pluripotency medium.
(C) Low magnification image of hiPSC cultures in mTeSR Plus at adequate confluency to begin 2D induction (∼60%).
(D) Stage 1: Induction of 2D hiPSC colonies in Induction Medium (N2B27 basal medium with 3 μM CHIR 99021 and 40 ng/mL FGF2) at 48 h. By visual inspection, optimal colony induction is characterized by slight raised-edge character just as cells begin to migrate away from the colony border (yellow arrows). Inset is white light image of primed colonies just before generation of single cell suspensions.
(E) Stage 2: Transition to shaking culture in EMLOC Polarization Medium. Single cell suspensions are generated and transferred to low-adhesion 6-well plates (2 × 106 cells/well). Spontaneous aggregation (size range ∼50–100 μm) at 24 h. Low 5× magnification is shown (left) with high magnification image (right).
(F) Stage 3: Early EMLOC polarization in Cardiac Induction Medium with cardiac crescent formation (white arrows) at day 4 post-aggregation.
(G) Polarized EMLOCs with contractile chamber-like structures (white arrows) at low magnification (left) and high magnification (right).
(H) Contracting cardiogenic region by live-cell calcium imaging using Fluo-4 AM dye in day 7 EMLOC.
(I) Stage 4: maturation of EMLOC chamber-like structures (yellow arrows). Two fields are shown. Individual scale bars are provided for all images.