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. 2023 Jun 6;9:53. doi: 10.1038/s41421-023-00543-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic overview of the PSC differentiation strategy based on image-based ML for solving the variability in efficiency, using CM differentiation as an example. Upper panel, variation occurs at each step in the PSC differentiation process. Lower panel, based on the bright-field image-based ML, our strategy is applicable at different stages to reduce the variation, achieving high-efficiency CM induction. b Schematic overview of PSC-to-CM differentiation with small-molecule modulators of canonical Wnt signaling. The green arrows (indicating the titration of CHIR durations and concentrations at stage I) and the colored dots represent the checkpoints involving ML in our strategy. c Example time-lapse bright-field images and cTnT fluorescence result of cardiac differentiation for 10 days. Cells were cultured in a 24-well plate. The enlargement of the image in the white frame is shown in d. Scale bar, 1 mm. d Location and morphology of successfully and unsuccessfully differentiated cells in the whole differentiation process. 0 h, 24 h, and 72 h bright-field images were enhanced and shown in inverted color for highlighting the edge of the cell colony (white arrows and blue arrows indicate the cell-containing area and cell-free area, respectively). Scale bar, 1 mm. e Texture and morphological change of successfully differentiated cells from day 5 to day 12. The white arrow indicates the day 6 CPC with texture. Scale bar, 250 μm. f Line-to-line variability of cardiac differentiation efficiency. CHIR duration = 24 h. g Batch-to-batch variability of cardiac differentiation efficiency. iPS18-derived CM differentiation followed the same differentiation protocol but from different batches for 12 days (CHIR concentration = 6 μM, duration = 24 h). Scale bar, 1 mm. h Change of image local features under different CHIR doses in cardiac differentiation. The y-axis is the first linear discriminant (LD1) given by LDA. The dark lines and the light lines represent the mean and the standard deviation value, respectively.