Regulation of cardiac transcription factors in liver stem cells co-cultured with cardiomyocytes after minimizing CAMTA1 expression.

<p>Except for the control conditions (A and B) the stem cells were pre-transfected with specific siRNA 24 hrs before they were added to the co-culture with cardiomyocytes. A: control, naive stem cells in monoculture for 72 hrs. B: naive stem cells cultured for 24 hrs, then co-cultured with cardiomyocytes for 48 hrs. C: Stem cells pre-transfected with a scrambled siRNA 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. D: Stem cells were pre-transfected with a human pool of CAMTA1 siRNAs for 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. The graphs illustrate expression of the transcription factors CAMTA1, CAMTA2, Nk×2.5, Myocardin, cTnT and BetaMHC. A significant decrease in the expression of transcription factors Nkx2.5, Myocardin, cTnT and BetaMHC were measured when CAMTA1 expression was minimized. Note that no significant change in CAMTA2 expression was observed. The bars show mean ± SEM, *p<0.05.</p

Time course of RNA expression levels of cardiac transcription factors and contractile protein genes in hMSCs co-cultured with rat neonatal cardiomyocytes.

<p>Control levels of CAMTA1, RCAN1, GATA4, Nk×2.5, Mef2c, Tb×5, cTnT, and BetaMHC in the stem cells in monoculture at day 0 (0d), or co-cultured with cardiomyocytes for 2 days(2d) and 4 days (4d). The bars show mean ± SEM, *p<0.05. # denotes novel expression in Nkx2.5.</p

Regulation of expression of cardiac transcription factors following the minimization of CAMTA1 expression in hMSCs monocultures.

<p>Human MSCs were pre-transduced with specific lentiviral vectors 24 hrs before the up-regulation of CAMTA1 expression was induced with ionomycin. A: control, naive hMSCs grown in monoculture, B: hMSCs pre-transduced with a GFP-lentiviral vector, then stimulated with ionomycin. C: hMSCs pre-transduced with CAMTA1 shRNA lentiviral vector, then stimulated with ionomycin. The graphs illustrate expression of the transcription factors CAMTA1, Mef2C, and Gata4. Note condition B shows increased expression of Mef2C and Gata 4 with increased CAMTA1 expression. With minimization of CAMTA1 expression (condition C) expression of Mef2C and Gata4 was significantly decreased (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone-0038454-g002" target="_blank">Figure 2D</a> for CAMTA1 control). The bars show mean ± SEM, *p<0.05.</p

Bone marrow-derived MSCs in co-culture with cardiomyocytes.

<p>(A) A nascent cardiomyocyte derived from mMSCs of a βMHC-YFP genotype mouse at 8 days in co-culture with cardiomyocytes. The βMHC-YFP cell is pseudo colored green for visualization of the striations. Left panel shows novel endogenous expression of βMHC-YFP fluorescence along stress fibers and striations (magnified in insert). Middle panel shows surrounding rat cardiomyocyte. Merged images in right panel. (B) GFP-hMSCs co-cultured with cardiomyocytes for 16 days and immunostained for α-actinin or troponin T (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454-MullerBorer1" target="_blank">[23]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454.s003" target="_blank">Supporting Information S1</a>). Acquisition of a differentiated cardiac phenotype is demonstrated in the inserts where cardiomyocyte striations are visible. Nuclear DAPI in blue. Scale bar = 20 µm.</p

Protein localization by immunocytochemistry in hMSCs co-cultured with cardiomyocytes for 48 hours.

<p>Left panels show dsRed-hMSCs, middle panels show CAMTA1 or NFAT protein localization. Right panels show merged images. (A) Novel expression of CAMTA1 protein is demonstrated in the nucleus of a dsRed hMSCs co-cultured with rat cardiomyocytes (arrow in middle panel). Rat cardiomyocytes (small nuclei in the background) a priori express CAMTA1. Note the larger human nucleus of the hMSCs compared to the size of the rat nuclei in the background. (B) Novel NFAT2c protein expression in an hMSC co-cultured with cardiomyocytes. NFAT2c is localized throughout the hMSC including the nucleus of the hMSC. NFAT2c was not detected in the nucleus in naïve hMSCs.</p