Fig 1: Differentiation of transduced cells into iCMs. (A) Seven days after transduction, morphology of MSCmiR-1 and MSCmiR-1/Myocd groups became spindle-shaped or star-shaped. Twenty-one days after transduction, the cells became more differentiated and matured and short spindle-shaped or polygonal cells were observed, similar to cardiomyocytes. (B) qRT-PCR analysis was performed for cardiac specific markers of Tnnt2, Nkx2-5, and Gata4. Expression of markers was compared in MSCmiR-1 and MSCmiR-1/Myocd groups with MSCnull as a negative control. The results showed that expression of Tnnt2, Nkx2-5, and Gata4 was increased significantly in MSCmiR-1/Myocd group in comparison with other groups, on days 7, 14, and 21 after transduction. * P < 0.05, ** P < 0.01, *** P < 0.001; Transcript value was shown as mean±SD. (C) ICC analysis detected cTnT, Nkx2-5, and GATA4 markers in MSCmiR-1 and MSCmiR-1/Myocd groups on day 21. Nucleus staining was conducted by DAPI stain. An Olympus fluorescence microscope was used to record images.
Fig 2: qRT PCR, histological studies and immunohistochemical staining of heart tissues after patch implantation. (A) qRT-PCR assessment of MI zone tissue in the intervention and control groups. (B) Histological analysis showed that myocytes in iCM/P group had regularly structure and red blood cells were observed, while in MI group, inflammatory cells infiltrated in MI zone and large area of fibrosis was detectable. C) Thirty days after MI induction, IHC staining was performed for NKX2-5, GATA4, and cTnT markers of MI zone tissue. The images showed that the immunostained cells in the iCM/P group were more than the other groups. Encapsulated cells of iCM/P and MSC/P groups were labeled with DiI stain for tracking in rats heart tissues.
Fig 3: Differentiation of transduced cells into iCM after encapsulation in the CS/CO hydrogel. Three days after co-transduction of MSCs with miR-1 and Myocd lentiviruses, the infected cells were trypsinized and embedded in the CS/CO hydrogel. (A) Expression of Tnnt2, Nkx2-5, and Gata4, and was evaluated by qRT-PCR in 2D and 3D cell cultures (CS/CO hydrogel). Expression of markers was increased significantly in the 3D cell culture in comparison with the 2D cell culture. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; Transcript value was shown as mean±SD. (B) Immunofluorescence staining of transduced cells 21 days after transduction showed that expression of the markers was enhanced in 3D culture. iCMs: Induced cardiomyocytes, CS/CO: Chitosan/collagen, 3D culture: Three dimensional culture.
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