Activin A Modulates CRIPTO-1/HNF4α⁺ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

Figure 1

Characterization of the embryoid body- (EB-) based cardiac differentiation protocol. Characterization was performed on human embryonic stem cell-derived cardiomyocytes (ESC-CMs) without the addition of Activin A (ActA) during the early stage of differentiation (control condition). (a) Schematic representation of the EB-based cardiac differentiation protocol. (b) Expression profiles for pluripotency (OCT4 and NANOG), mesodermal (BRACH), cardiac progenitor (NKX2.5 and GATA4), and late-stage CM (cMyHC, TNNT2, TNNI3, and HCN4) markers, monitored during 19 days of differentiation. (c) IF analyses of dissociated EBs at day 20 of differentiation, showing areas of connected CMs (cMyHC; red) expressing connexin 43 (CX43; green). Nuclei were stained with Hoechst (blue). (d) Flow cytometry analyses of cMyHC⁺ CMs at day 20 of differentiation. Representative example of three independent experiments. (e) Western blot analyses quantifying the cMyHC protein levels of undifferentiated ESCs and CMs after 20 days of differentiation, normalized by GAPDH. (f) -Adrenergic response of CMs to isoprenaline (1 μM; isoproterenol), triggered at day 20 of differentiation. (g) Time course of intracellular Ca²⁺ handling at day 20 of differentiation, using the Ca²⁺-sensitive fluorescent indicator Fluo-4 (2.5 μM), monitored by confocal microscopy. (h) Whole-cell Na⁺ recording, accessed by applying 500 ms voltage pulses to potentials between −120 and +10 mV in 5 mV increments from a holding potential of −100 mV at 0.5 Hz. RT-QPCR data are represented as , normalized for the housekeeping genes GAPDH, HPRT, and RPL13a. Data are representative of three independent experiments and values are expressed as mean ± standard error of the mean (SEM). Significant difference is versus control and indicated as : ∗∗∗. Scale bar = 100 μm.