Fig 2: Morphological characteristics, differentiation induction and phenotypic identification of BM-MSCs. (A) Bright field image of third-passage BM-MSCs following transduction with HO-1, growing as long spindle-shapes with a whirlpool or paralleled pattern (magnification, ×100). (B) Adipogenic induction (magnification, ×200), the arrow presents the orange lipid droplets in the cytoplasm after staining with Oil Red O. (C) Osteogenic induction (magnification, ×200), the arrow indicates the black calcium salt deposit after von Kossa staining. (D) Fluorescent image of third-passage BM-MSCs following transduction with HO-1, showing that ~85% of the cells are transduced with HO-1 (emitting green fluorescence; magnification, ×100). (E) 98.3% of the cells CD29+CD34- cells. (F) 97.6% of the cells are CD90+CD45- cells. (G) 92.3% of the cells are RT1A+RT1B- cells. BM-MSCs, bone marrow mesenchymal stem cells; HO-1; heme oxygenase-1.

Fig 3: Culture and identification of rat BMSCs. (A–C) The positive markers were used to differentiate the stem cells from other cells. The positive rates of CD29, CD90 and CD106 were 92.5%, 100% and 91.1%, respectively. Red line represents the antibody to be tested, and blue or orange line represents the homotypic control. (D,E) The negative markers CD11b and CD45 were used to distinguish mesenchymal stem cells from hematopoietic stem cells. The positive rates of CD11b and CD45 were 0.080% and 0.030%, respectively. Red line represents the antibody to be tested, and blue line represents the homotypic control. (F) Adipogenic induction and Oil Red O staining; (G) Osteogenic induction and Alizarin Red S staining.

Fig 4: Representative images of flow cytometry analyses for rat brown adipose-derived cells. Brown adipose-derived cells are partly positive for CD-29, CD90, and CD133, while negative for CD45 and CD34. (a) Control; (b) Flow cytometry analysis for surface marker CD90; (c) Flow cytometry analysis for surface marker CD29 and CD133; (d) Flow cytometry analysis for surface marker CD45; (e) Flow cytometry analysis for surface marker CD34.

Fig 5: Characterization of MSCs derived from rat bone marrow and transfection efficiency of MSCs with pEF1/His C::IGF-1::EGFP. (A) Expression levels of surface markers in the isolated MSCs were measured by flow cytometry for CD29-FITC, CD54-PE, CD90-FITC, CD45-PE and CD49d-FITC. The red histograms represent staining with respective surface marker antibodies and the black histograms represent staining with isotype controls. (B) Prior to FACS, the transfection efficiency of MSCs with pEF1/His C::IGF-1::EGFP and the nonviral carrier PAM-ABP was ~20% (P4). (C) Following FACS, transfected MSCs were collected and the purity was increased to 95.1%. Therefore, approximately all MSCs expressed a green fluorescent signal and IGF-1. CD, cluster of differentiation; EGFP, enhanced green fluorescence protein; IGF, insulin-like growth factor; MSC, mesenchymal stem cells; FACS, fluorescence-activated cell sorting cell sorting; FITC, fluorescein isothiocyanate; PE, phycoerythrin.