Figure 4.

The effects of EB age on post-disaggregation IPS-CM density and plating heterogeneity. (A) (i,ii) A grid-based scheme for determining cell density and plating heterogeneity in A- (red), B- (blue), and C- (black) areas of post-EB IPS-CM populations. In this 7 × 7 grid system each B-area corresponds to 1/49th of an A-area and each C-area represents 1/49th of a B-area (see Section Materials and Methods). (ii) Representative overlays of DAPI-stained and brightfield images acquired from A-areas of IPS-CM populations following EB disaggregation EBs at 0.5 week intervals. The arrow depicts a typical CM cluster that would be selected for Ca²⁺ image analysis throughout this study. (iii) A C-area image of troponin-T immunostaining (red) in an IPS-CM following the disaggregation of a 0.5 week EB maintained “on plate” for a further week. The nucleus is counter-stained with DAPI (blue). Arrow indicates troponin-T striation. (B,C) Scatterplots of cellular density in A- and B-areas, respectively, following disaggregation from EBs at 0.5-week intervals. Each point represents the analysis of a single area (n = 5–40). Data were fitted with least-squares polynomial function (red and blue lines for A- and B-areas, respectively) and tau and r²-values are given. (D) The plating heterogeneity of IPS-CM was determined from random A-areas (circle) or from B-areas (triangles) across the 4-week protocol. Data are means from n = 5–40 areas in each instance and are fitted with least-square polynomial function (A-area, blue; B-area, red) and r²-values are given. (E) The proportion of B-areas that were devoid of cells following post-EB disaggregation and seeding was determined. Data are plotted as means from n = 40 A-areas in each instance and were subject to linear regression (blue line) and the r²-value is given.