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

Monolayer cultures of highly purified ESCM.

<p>A) Bright field image of puromycin-resistant ESCM at D7+9. In this example, D7+1 EBs were collagenase treated and replated in the presence of puromycin for 3 days. Bar = 50 µm. B) Selected cells (D7+4) were stained with Hoechst 33342 (ii, iii and vi) and probed with antibodies to either α-actinin (i and iii) or cTnT (iv). Magnified views (inset) show individually stained CM. Bar = 100 µm (Insert = 30 µm). C) Puromycin resistant cells (D7+3) were probed for cTnT and analyzed by flow cytometry. The results showed that >98% of the ungated cells in the monolayer culture were positive for cTnT. Insets show forward and side scatter plots of analyzed cells (linear scale). D) Transcripts to cardiac-associated Nkx2.5, Gata4, α-MHC, Mlc2a and Mlc2v were highly abundant, but those to Sox1, Sox17, MyoD and von Willebrand factor (vWF) were not detected in any of the pooled RNA samples from monolayer cultures. E) QRT-PCR analysis of gene transcripts differentially expressed during CM maturation, showing a significant increase in calsequestrin and decrease of atrial natriuretic factor (ANF) as a function of time. Slow skeletal (ss) isoforms of TnI were also temporally replaced by cTnT.</p

Knockdown of Rb in proliferating CM does not induce apoptosis.

<p>A) CM were stained with cTnT and Hoechst 33342 at two time points, D2 and 5, after transfection with siRNA-Rb. The appearance of condensed or fragmented nuclei and activated caspase 3, as an index of apoptosis, was then assessed. In these experiments, no altered nuclear fragmentation or condensation was observed among any of the groups, following KD of Rb. Magnified views (inset) show nuclei stained with Hoechst 33324 without characteristics of apoptosis. Bar = 50 µm (Insert = 10 µm). B) Western blot showing phosphocaspase-3, which was used as an indirect measure of apoptosis.</p

Regulators of G1 cell cycle progression are dynamically regulated in early ESCM.

<p>A) QRT-PCR analysis of activator and repressor E2F and B-Myb associated transcripts. (*, P<0.05 vs D7+3, §, P<0.05 vs D7+6). B) Relative abundance (qRT-PCR) of pocket proteins (Rb, p107, p130) as a function of time (*, P<0.05 vs D7+3, §, P<0.05 vs D7+6). C) Western showing the abundance of pocket proteins in ESCM and the phosphorylation state of Rb (*, P<0.0005 vs D7+3, §, P<0.005 vs D7+3, ¶, P<0.05 s D7+6) . Relative abundance of hypo- (pRb) to hyperphosphorylated (ppRb) is shown graphically.</p

Knockdown of Rb alters the expression of factors implicated in the G1 to S phase transition.

<p>A) Image of purified ESCM transfected with siGlo RNA showing that >90% of the puromycin-resistant cells were transfected. Bar = 50 µm (Insert = 10 µm). B) QRT-PCR analysis of Rb, p107 and p130 mRNA in purified ESCM transfected with siRNAs against Rb (*, P<0.05 vs control, §, P<0.05 vs non-target). C) Western blot images showing Rb, p107 and p130, following transfection with siRNA-Rb8. Loading control (actin) image was altered from a bitmap image, due to the loss of the tiff file. D) QRT-PCR analysis of E2F1-E2F5 and B-Myb, following KD of Rb (*, P<0.05 vs control, §, P<0.05 vs nontargeting, ¶, P<0.005 vs control). E) Percentage of BrdU labeled CM (cTnT positive) after KD of Rb in puromycin selected cells (b, *, P<0.0005 vs control, §, P<0.0005 vs nontargeting). F) Cell cycle distribution (DNA content of PI-stained nuclei) of cells after KD of Rb relative to controls (*, P<0.0005 vs control, §, P<0.05 vs nontargeting, ¶, P<0.005 vs Control, ∫, P<0.005 vs nontargeting). g) Graph showing CM numbers following siRNA-Rb mediated KD of Rb in puromycin-resistant cells (*, P<0.0005 versus control, §, P<0.0005 versus nontargeting).</p

Novel FFA1 (GPR40) agonists containing spirocyclic periphery: polar azine periphery as a driver of potency

<p>A series of nine compounds based on 3-[4-(benzyloxy)phenyl]propanoic acid core containing a 1-oxa-9-azaspiro[5.5]undecane periphery was designed, synthesized and evaluated as free fatty acid 1 (FFA1 or GPR40) agonists. The spirocyclic appendages included in these compounds were inspired by LY2881835, Eli Lilly’s advanced drug candidate for type II diabetes mellitus that was in phase I clinical trials. These polar spirocyclic, fully saturated appendages (that are themselves uncharacteristic of the known FFA1 ligand space) were further decorated with diverse polar groups (such as basic heterocycles or secondary amides). To our surprise, while seven of nine compounds were found to be inactive (likely due to the decrease in lipophilicity, which is known to be detrimental to FFA1 ligand affinity), two compounds containing 2-pyridyloxy and 2-pyrimidinyloxy groups were found to have EC₅₀ of 1.621 and 0.904 µM, respectively. This result is significant in the context of the worldwide quest for more polar FFA1 agonists, which would be devoid of liver toxicity effects earlier observed for a FFA1 agonist fasiglifam (TAk-875) in clinical studies.</p