Fig 1.
BM-MSCs acquire specific cardiomyocyte markers after co-culture with rat embryonic cardiomyocytes (RECs) but not when co-cultured with cells derived from other embryonic tissues.
(A) Schematic for experimental design to study cardiomyocyte differentiation of MSCs by co-culture with RECs. BM-MSCs were isolated from genetically modified mice that express α-MHC promoter-driven GFP. (B) RT-PCR primers were designed to specifically detect the expression of cardiac specific genes in mouse-derived mRNA (mHe) and do not amplify rat mRNA sequences (RECs). (C) Quantitative real time RT-PCR was performed to determine the expression of cardiac specific genes in mouse BM-MSCs after co-culture with RECs for 5 days. Data were normalized against the reference gene GAPDH and presented as relative units taking the expression in mouse heart as 1 unit. Data represent mean±SD of five independent experiments. *p<0.0001 between CC and MSCs groups derived from one-way ANOVA after Tukey's multiple comparisons test. (D) Representative images of BM-MSCs at 5 days of co-culture (original magnification 200x). MSCs were identified in the co-culture by Collagen type IV (Col IV) immunostaining. Cells undergoing cardiomyocyte differentiation express GFP (green cells). (E) α-MHC promoter activity was calculated as the percentage of Col IV-positive cells expressing GFP. Data represent mean±SD of four independent experiments. *p<0.0001 between groups derived from unpaired t test. (F) RT-PCR assay for the expression of cardiac specific genes in mice BM-MSCs after co-culture with rat embryonic cells from lung (RELu), kidney (REKi) and liver (RELi). The results of three independent co-culture experiments are shown. Abbreviations: α-MHC, alpha-myosin heavy chain; ANF, atrial natriuretic factor; CA, cardiac actin; CC, co-culture; Col IV, collagen type IV; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mHe, mouse heart; RECs, rat embryonic cardiomyocytes.
Fig 2.
Changes in the expression of the pluripotency factors OCT4, SOX2 and NANOG in BM-MSCs after co-culture.
(A) RT-PCR primers were designed to specifically detect gene expression of pluripotency factors in mouse mRNA (ESCs) without amplification of rat mRNA sequences (RECs). (B) Expression of pluripotency genes in untreated BM-MSCs and MSCs after co-culture with RECs as determined by qRT-PCR. Data were normalized against the reference gene GAPDH and presented as relative units taking the expression in untreated MSCs as 1 unit. Data represent mean±SD of five independent experiments. *p<0.001 between groups derived from unpaired t test. (C) Western blot showing changes in the expression of OCT4, SOX2 and NANOG in MSCs after the co-culture (n = 3). Total protein extract from ESC was used as positive control. Detection of β-Tub was used as loading control. Abbreviations: CC, co-culture; ESC, mouse embryonic stem cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MW, molecular weight marker; RECs, rat embryonic cardiomyocytes.
Fig 3.
The frequency of OCT4 expression in BM-MSCs decreases after co-culture with RECs as assayed by flow cytometry.
(A) BM-MSCs were stained with an APC-conjugated anti-mouse CD44 antibody. More than 98% of the cells are positive for this marker. Gray dotted line, isotype control; black line, antigen staining. (B, C) The CD44 antibody does not cross react with RECs and therefore was used to distinguish mouse from rat cells in the co-cultures. (D) OCT4 expression analyzed on the CD44+ gated population in untreated MSCs and MSCs after co-culture with RECs. Representative histograms. (E) Changes in the frequency of OCT4+ cells after the co-culture. Data represent mean±SD of four independent experiments. p value between groups derived from unpaired t test. Abbreviations: CC, co-culture; RECs, rat embryonic cardiomyocytes.
Fig 4.
MSCs undergoing cardiomyocyte differentiation (GFP+ cells) lose expression of pluripotency factor, OCT4.
(A) Schematic for GFP+ cell sorting after 5 days of co-culture with RECs. Representative dot plot illustrating forward-scatter vs GFP flow cytometry analysis on the GFP+/CD44+ gated population. (B,C) GFP+ sorted cells express the cardiac-specific protein troponin-T (TnT), but retained the expression of the stromal marker collagen type IV (Col IV). Images are representative of three independent experiments (original magnification 400x). (D) Immunocytochemical analysis of OCT4 expression in sorted GFP+ and GFP- MSCs after 5 days of co-culture. Images are representative of three independent experiments (original magnification 400x). (E) Bisulfite sequencing analysis of the OCT4 promoter in sorted GFP+ and GFP- MSCs after 5 days of co-culture. Each horizontal row of circles represents an individual sequencing reaction for a given amplicon. Open and closed circles indicate unmethylated and methylated CpGs, respectively. The overall percentage of methylation is noted to the right of each panel.
Fig 5.
OCT4 expression is required for partial cardiomyocyte differentiation of MSCs during co-culture with RECs.
BM-MSCs were incubated for 24 h with OCT4 siRNA (siOCT4) or a scrambled control siRNA (siScr) and transfected cells were then selected using puromycin. (A) Quantitative realtime PCR assay for expression of OCT4, SOX2 and NANOG in OCT4 siRNA silenced (siOCT4) and control siRNA MSCs (siScr). Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level in untreated MSCs. Data represent mean±SD of four independent experiments. *p<0.01 and #p<0.001 between groups derived from unpaired t test. (B) Western blot analysis of the OCT4 protein in OCT4 siRNA silenced (siOCT4) and control siRNA MSCs (siScr) (n = 3). (C) Expression of cardiac specific genes in siOCT4 vs. siScr MSCs after co-culture with RECs for 5 days as assessed by qRT-PCR. Data were normalized against the reference gene GAPDH and presented as relative units taking the expression in mouse heart as 1 unit. Data represent mean±SD of four independent experiments. *p<0.05 and #p<0.0001 between siOCT4 and siScr MSCs groups derived from one-way ANOVA after Tukey's multiple comparisons test. (D) Cardiomyocyte differentiation frequency in siOCT4 vs. siScr MSCs after co-culture with RECs for 5 days. α-MHC promoter activity was calculated as the percentage of Col IV-positive cells expressing GFP. Data represent mean±SD of four independent experiments. *p<0.001 between groups derived from unpaired t test. Abbreviations: α-MHC, alpha-myosin heavy chain; ANF, atrial natriuretic factor; β-Tub, β-tubulin; CA, cardiac actin; CC, co-culture; Col IV, collagen type IV; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mHe, mouse heart; RECs, rat embryonic cardiomyocytes.
Fig 6.
OCT4 expression is required for interaction of MSCs with the cardiac microenvironment.
A schematic depicting the main mechanisms proposed in the present study. (1) MSCs have a basal expression of OCT4 and release several growth factors and cytokines under normal conditions. (2) MSCs interact with the cardiac microenvironment and partially differentiate into cardiomyocytes, by an indirect mechanism. (3) As a result of the interaction with the cardiac microenvironment, MSCs de-differentiate with a net gain in OCT4 expression. (4) De-differentiated MSCs express higher levels of OCT4 and can start the differentiation process into cardiomyocytes. (5) MSCs acquire a partial cardiomyocyte phenotype that modulates the paracrine effect and improves their cardiac regenerative potential. (6) Full cardiomyocyte differentiation of MSCs to generate completely mature cardiomyocytes was not observed. Abbreviations: ESC, embryonic stem cells; MSCs, mesenchymal stromal cells.