Figure 7. TLR3 agonists enhance maturation of miR combo reprogrammed cardiomyocytes.

(A–C) Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with vehicle or the TLR3 agonist Poly(I:C) LMW (low molecular weight Poly(I:C)) for a further 4 days. After incubation with the TLR3 agonist, cells were cultured in normal growth media for a further 10 days.

(A) RNA levels of the cardiomyocyte structural proteins Myh6 (αmyosin heavy chain), Actn2 (αsarcomeric actinin) and Tnni3 (cardiac troponin-I) was determined by qPCR. N=5–14.

(B) Cells were fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). N=6 independent experiments. Scale bar 50 microns. Inset pictures are at 5x magnification to show sarcomere structure.

(C) Quantification of immunostaining shown in B. Cells expressing Actn2 were counted and expressed as a percentage of the total cell population.

(D) Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with differentiation media (DMEM + 2%FBS + ITS + AA) and the TLR3 agonist Poly(I:C) LMW for the indicated times. Fourteen days after the transfection, the numbers of beating colonies were counted. N=4 independent experiments. Data represented as Mean ± SEM. *Comparisons made between vehicle and TLR3 agonist for each group ***P<0.001, **P<0.01, *P<0.05. †Comparisons made between miR combo and negmiR for each group †††P<0.001, ††P<0.01, †P<0.05.