Fig 1.
Schematic of GiWi and co-culture method used to initiate cardiac differentiation of iPS cells.
(A) For differentiation by GiWi, iPS cells were seeded onto Matrigel-coated plates and cultured to ~90% confluence in mTeSR1. The basal media was switched to RPMI/B27 at days 0–7, with addition of CHIR-99021 at days 0–1, and IWP-2 at days 3–5. Thereafter, the basal media was switched RPMI/B27/insulin. (B) For co-culture differentiation, GFP+ve iPS cells (AICS11 or AICS16) were similarly cultured to ~90% confluence in mTeSR1. At day 0, iCMs derived from non-labeled GiWi-differentiated iPS cells were dissociated and added to GFP+ve iPS cells. Cells are maintained in RPMI/B27 at days 0–7 (without GiWi), and subsequently in RPMI/B27/insulin. Black arrows indicate key steps, while gray arrows indicate the frequency of culture media replacement.
Fig 2.
AICS11 and AICS16 iPS cells co-cultured IMR90 iCMs exhibit spontaneous self-contractions.
(A) DIC image of pre-culture on day 23 (scale bar: 500μm), red box shows magnified overlay of DIC and GFP images (scale bar: 100μm). Kymograph of contracting cluster indicated by red arrow (scale bar: 50μm). (B-D) Co-cultures were dissociated and reseeded at low cell density for imaging and kymograph analysis of isolated cells. Kymogram traces are for the indicated colored arrows. In (D), a non-contractile GFP+ve cell (blue arrow) is shown in proximity to a GFP-ve cell exhibiting self-contractions (red arrow). Scale bars: 25μm in overlays; 5μm in kymographs. Cells are AICS16 for A; AICS11 for B,C,D.
Fig 3.
Staining of AICS11 (TOM20-GFP) cells for sarcomeric α-actinin.
AICS11 cells were differentiated using (A) GiWi protocol or (B) inductive co-culture with iCMs, and (C) basal media change alone (absent differentiation factors). Bottom panels show a magnified image of α-actinin staining for the area bounded by white rectangles. Scale bars are 50um for top panels and 5um for bottom panels.
Fig 4.
Staining of AICS16 (β-actin-GFP) cells for sarcomeric α-actinin.
AICS16 cells were differentiated using (A) GiWi protocol or (B) inductive co-culture with iCMs, and (C) basal media change alone (absent differentiation factors). Bottom panels show a magnified image of α-actinin staining for the area bounded by white rectangles. Scale bars are 50um for top panels and 5um for bottom panels.
Fig 5.
Cardiomyocyte differentiation efficiency of AICS11 co-cultured with iCMs evaluated by flow cytometry.
AICS11 were either co-cultured with IMR90-iCMs, or subjected to media change only as a control. Additional controls include non-differentiated iPS or GiWi-differentiated AICS11 or IMR90 cells. Total cells were harvested and immuno-stained for the cardiomyocyte markers (A) cTnT and (B) a-actinin, and analyzed by flow cytometry in conjunction with TOM20-GFP to distinguish AICS11 cells. As shown are representative flow plots for an experiment performed in triplicates. Calculation of cardiomyocyte differentiation efficiency for (C) cTnT and (D) a-actinin. Green bars indicate cells gated for GFP only, where % = Q2/(Q2+Q3)*100, while grey bars indicate total cells, where % = (Q1+Q2)/(Q1+Q2+Q3+Q4)*100. P-value for all pairwise comparison is ≤0.0001, except otherwise indicated, where ns is non-significant, and ** is <0.002.
Fig 6.
Cardiomyocyte differentiation efficiency of AICS16 co-cultured with iCMs evaluated by flow cytometry.
Description for this experiment is identical to that for Fig 5, but with AICS16 cells (expressing GFP-b-actin) in place of AICS11. P-value for all pairwise comparison is ≤0.0001, except otherwise indicated, where ** is <0.003.