Figure 1: The CHIR-to-volume ratio during the first 24 h dictates differentiation outcome.

(a) Schematic of expansion and subsequent differentiation. The medium volume of 1–3 ml and consequently the BCD is only modulated between day 0–1. (b) Representative images of the differentiation on day 10 using the NKX2.5-GFP cell line. The cornerstone conditions are marked by a coloured rectangle. Scale bar, 1 mm. (c) Analysis of NKX2.5-GFP⁺ on day 10 of the four cornerstone conditions, n=11 independent experiments. The mean is shown as black line. *P<0.01, evaluated by one-way analysis of variance with post hoc Bonferroni analysis. (d) Representative histograms for NKX2.5-GFP⁺ and staining against structural cardiac markers on day 10 obtained by flow cytometry. (e) Schematic of experiment (left) and corresponding NKX2.5-GFP⁺ on day 10 (right) in standard conditions using identical cell numbers in different volumes (A) and in identical volume with adapted cell numbers for static as well as agitated conditions (B). Bars represent mean±s.e.m. of n=3 independent experiments. (f) Representative images of NKX2.5-GFP expression on day 10 of differentiation performed in 2D monolayer cultures. Scale bar, 1 mm. See also Supplementary Fig. 1. NS, not significant.