Fig. 4.

Cell-state diversity at the tipping point predicts differentiation efficiency. (A) Perturbation scheme to influence the M–En branching event on day 3. In total, we tested 15 BMP4/GSK3 inhibitor combinations highlighted with a unique color or number in a circle across all panels herein. (B) Dynamics of the cKIT distribution during early differentiation reflect the effect of each small molecule combination. We observed different cKIT distributions on days 2 and 3 for each treatment (flow cytometry data shown in Fig. S8), which inform predictions about protocol efficiency as measured by the proportion of cTNNT2+ cells—the most commonly used molecules to show the cardiac fate—on day 28 (shown in striped boxes). (C) Projections of the 100-cell pool cKIT^High, cKIT^Medium, and cKIT^Low gene expression profiles onto the diffusion map (DM). The single-cell gene expression profiles are shown in gray, and combination-specific, population-specific pooled gene expression vectors are represented with the corresponding colors from A. The position in the trajectory at day 2 as well as the variable distribution of cKIT outlier fractions (shown as numbers in boxes above histograms) can explain huge variation in the efficiencies of the protocols (measured as cTNNT2+ cells at day 28). (D) Random Forest analysis to identify transcripts or cell subpopulations that predict high- vs. low-efficiency protocols. We classified every combination that produces >60% iCMs as high efficiency (dark blue), whereas any combination with efficiency <60% was classified as low efficiency (orange). (E) The cKIT^High, cKIT^Medium, and cKIT^Low concatenated gene expression vectors outperform the cKIT^Medium vector classification. HAND1 gene expression in cKIT^High and cKIT^Low populations discriminates between three different combinations with different efficiencies (marked by asterisks), whereas SOX17 and HAND1 gene expression in cKIT^Medium population is the same. (F) Correlation plots for signaling molecules and combination class (low vs. high efficiency). Low-efficiency protocols are marked by lower dosages of exogenous BMP4 (<2.5 μM), but it seems that there is no correlation between exogenous administration and endogenous BMP4 levels (measured as gene expression; no correlation). However, higher dosages of exogenous BMP4 are highly correlated with the expression levels of DKK1 and Wnt5B in the cKIT^Low population that will commit to the cardiac cell fate. Again, cKIT^High or cKIT^Low gene expression vectors perform better than cKIT^Medium.