Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

Puromycin selected ESCM proliferate <i>in vitro</i>.

<p>A) Images of CM in various stages of mitosis. Puromycin-resistant cells were stained with Hoechst 33342 (blue), and probed with antibodies specific to cTnT (red; i–iii, green; iv–vi) and phospho-H3 Histone (red; iv-vi). In examples i-iii, condensed chromatin, metaphase and telophase were observed at D7+3–4. In iv–vii, mitotic activity was confirmed by staining with an antibody to phospho-H3 Histone, which is only phosphorylated at Ser10 during mitosis. Mitotic nuclei are pink (red+blue). Bar = 10 µm. B) Graph showing that CM numbers increase by 2.4±0.2-fold between D7+3 and 7+6 (*, P<0.005).</p

Early stage CM actively progress through the cell cycle.

<p>A) BrdU positive (green, S phase), cTnT positive (red) cells were routinely observed at early time points after selection, but only rarely at late time points. Bar = 50 µm. B) Graph depicting the percentage of BrdU labeled CM as a function of time (*, P<0.005 vs D7+3, §, P<0.05 vs D7+6, ¶, P<0.005 vs D7+6, ∫, P<0.05 vs D7+9). C) Graph showing the percentage of binuclear CM stained with both cTnT and Hoechst 33324 (Bar = 50 µm) between D7+3 and 7+15 (*, P<0.005 vs D7+3, §, P<0.05 vs D7+6, ¶, P<0.005 vs D7+6, ∫, P<0.05 vs D7+9). D) DNA content was measured on PI-labelled fixed cells by flow cytometry, and the relative positions of G0/G1, S and G2/M phase cells are shown. The graph indicates the number of cells in each phase of the cell cycle as a function of time (*, P<0.05 vs D7+3, §, P<0.05 vs D7+6, ¶, P<0.05 vs D7+3). E) DNA content analysis of 120 hour nocodazole-treated cells and percentage of cells in each phase of the cell cycle. *, P<0.005 versus control.</p