Fig. 4: Enzymatic cross-correction of unedited cardiomyocytes by gene-corrected cardiomyocytes.

a Schematic of enzymatic cross-correction experiments using gene-corrected cardiomyocytes. Unedited iPSC-CMs (red) were supplied media without glucose for 24 h (orange). After 24 h, media was replaced with media (pink) that had previously been exposed to corrected cell lines (blue) or supplemented with rhGAA. 24–96 hours after replacement, unedited cells were stained with LysoSensor and imaged using confocal microscopy for dye intensity. b Quantification of LysoSensor intensity in cross-corrected lines 96 h post media exchange. Each triangle represents a corrected cell identified using CellProfiler. After 96 h of daily media changes or supplementing with rhGAA, all conditions had a significant increase in dye intensity over control conditions. Unedited cells were modified to express histone 2B (H2B)-mCherry to facilitate imaging of the nuclei in these assays. See also Supplementary Fig. 7. (***p < 10⁻¹⁵, n = 262, 201, 261, 249, 135, and 302 independent lysozymes respectively per condition, as detailed in Supplementary Table 7, two-tailed t-test, α = 0.05, heteroscedastic; mean ± s.d). c Representative images of unedited iPSC-CMs stained with LysoSensor in media from unedited and double-corrected iPSC-CMs. d Representative images of LAMP1 staining in unedited, single corrected, double-corrected cells and control PSC-CM and unedited iPSC-CM treated with rhGAA. (scale bars: 10 µm). Select enlarged LAMP1-positive lysosomes are identified by white arrowheads.