Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells

Figures

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  Fig 1. Expression of markers of interest during differentiation of mouse ESCs Ccbe1 in cardiac progenitors isolated from differentiating mouse ESCs and embryos at E9.5. (A-F) Samples were collected from undifferentiated cells (Und) and at days 2, 4, 6, 8 and 10 of differentiating RG mouse ESCs. Expression is presented as fold change relative to undifferentiated cells. Data presented as the mean + SEM of two biological replicates in technical qPCR triplicates. (G-M) Ccbe1 expression was analyzed in isolated populations, defined as FHF G+R- population, SHF G+R+ and G-R+ populations and control G-R- population at (G) day 6, (H) day 8 and (I) day 11 from differentiating mouse ESCs, and from (K) E9.5 mouse embryos. Expression is represented as fold change relative to the control G-R-population. (J) Analysis of Ccbe1 expression in the control G-R- population at day 6, 8 and 11 relative to the expression at day 6. The identity of the sorted populations at day 6 was confirmed by analyzing the expression of (L) Ist1and (M) Nkx2.5. Mean + SEM of two biological replicates.

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  Fig 2. Expression of Ccbe1 during differentiation of Ccbe1 knockdown mouse ESC lines. Transgenic mouse ESC lines expressing control scrambled and Ccbe1-targeting shRNAs (Clone 1 and 2) were generated using commercially available shRNA control and two Ccbe1-targeted shRNA lentiviral plasmids. Selected clones of the transgenic Ccbe1KD ESC lines and the control mouse ESC line were allowed to differentiate. Samples were collected from undifferentiated cells (D0) and at days 2 (D2), 4 (D4), 6 (D6), 8 (D8) and 10 (D10) of differentiating clones. Expression is presented as fold change relative to the control of each day. Data presented as the mean + SEM of three biological replicates in technical qPCR triplicates.

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  Fig 3. Analysis of expression of germ layer markers by immunofluorescence at day 10 of differentiation. shRNA control, Clone 1 and Clone 2 were let to differentiate until day 10. Differentiation into the three germ layers was analyzed by immunofluorescence of Alpha-fetoprotein (Endoderm), β-tubulin III (Ectoderm) and alpha smooth muscle actin (Mesoderm). Negative controls were only incubated with secondary antibody. Scale bar: 50 μm.

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  Fig 4. Ccbe1 knockdown leads to reduced cardiac mesoderm formation from differentiating mouse ESCs. (A) qPCR analysis of two individual Ccbe1 KD ESC clones (Clone 1 and Clone 2) compared to SH Control at Day 4 of the mesoderm marker brachyury (BraT) and cardiac precursor markers Mesp1, Isl1, and Nkx2.5; and at Day 6 of the cardiac markers Isl1, Nkx2.5, cTnt and αMhc. (B) qPCR analysis between Day 6 and 10 of the cardiomyocyte marker cTnT. All samples are relative to the control at Day 6. Mean ± SEM of three biological replicates in technical qPCR triplicates; unpaired t-test relative to Control; statistical significance *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001. (C) Plated EBs were checked every day for beating and then analyzed at day 10 of differentiation. Beating foci were counted under the microscope. Mean ± SEM of at least three biological replicates; unpaired t-test relative to Control; statistical significance *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

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  Fig 5. Ccbe1 loss-of-function leads to smaller embryoid bodies. (A) Phase-contrast micrographs of Ccbe1 KD ESC-derived embryoid bodies and control at days 3, 4 and 5 of differentiation. (B) Corresponding diameter increase of embryoid bodies at days 4 and 5 of differentiation (relative to day 3). (C) Cell proliferation was analyzed by labeling newly synthesized DNA using EdU incubation for 1.5 hours. Subsequent EdU labeling was done following the Click-iT EdU Alexa Fluor 488 Imaging Kit indications. Relative fluorescence intensity of cells was analyzed by flow cytometry. Minimum number of events: 20,000. (D) Quantification of immunolabelled CASP-3 positive cells analyzed by Flow cytometry at day 5 of differentiation.

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  Fig 6. Ccbe1 loss-of-function by neutralizing antibody also leads to smaller embryoid bodies. (A) Phase-contrast micrographs of WT ESCs derived embryoid bodies at days 3, 4 and 5 of differentiation with and without supplementation of 100ng/mL of CCBE1 antibody. Graph on the right side shows the corresponding diameter measurements of the embryoid bodies at day 5 of differentiation; unpaired t-test relative to non-supplemented group. (B) qPCR analysis of cardiac precursor markers Mesp1 and Isl1at day 4 of differentiation. Data represent the mean ± SEM of three biological replicates in technical qPCR triplicates; paired t-test relative to Control group; statistical significance *p<0.05, **p<0.01, ***p<0.001.