Supplement 1Characteristic and Multi-Differentiations of Mscs

Supplement 1Characteristic and Multi-differentiations of MSCs

Figure 1.A–F. Phenotype characterization of MSCs. Flow cytometric analysis showed that MSCs expressed CD90 (96.7%), CD29 (94.6%), CD34 (0.79%), and CD45 (0.84%).G.RT-PCRanalysis of CD105, CD90, CD166, Oct-4, Nang, CXCR4, c-Kit and CD34 gene expression in MSCs.Rat GAPDH served as aninternal control.H. RT-PCRanalysis of nkx2.5, GATA4, MHC,MHC, CD31, and MEF2Cgene expression in MSCs.Rat GAPDH served as aninternal control.Introduced differentiation of MSCs in specialculture conditionsin vitro. (I) After differentiation into osteocytes after von Kossa staining;(J) Afteradipogenicdifferentiation.

RT-PCR

Total RNA was isolated from MSCs. The amount and qualityof RNA samples were determined by measuring optical densityat 260 nm and analyzing 28S rRNA on an agarose gel, respectively.Total RNA (5 µg) was reverse-transcribed from an oligo(dT)primer using a first-strand synthesis kit (Stratagene Europe,Amsterdam, The Netherlands) in the conditions recommended bythe supplier. Reaction conditions were 10 mM Tris·HCl, 50 mM KCl,0.1% Triton X-100, 250 µM 2-deoxynucleotide 5'-triphosphate,1.5 mM MgCl2; 1 U of Taq DNA polymerase (Promega);and 0.5 µM each of forward and reverse primers (Table 1)in a 20-µl volume. PCR was performed at 95°C for4 min, followed by 25-33 cycles of 94°C for 45 s, 56°Cfor 40 s, and 72°C for 40 s. GAPDH was used as a control for the quality of the cDNA foreach PCR reaction.PCR products were separatedby performing electrophoresis in agarose gels containing ethidiumbromide and visualized under UV light. All PCR products weresequenced to verify their identity.

Reference:

1.Oyama T, Nagai T, Wada H, Naito AT, Matsuura K, Iwanaga K, Takahashi T, GotoM, Mikami Y, Yasuda N, Akazawa H, Uezumi A, Takeda S, Komuro I. Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo.J Cell Biol. 2007;176(3):329-41.

2.Yaghoobi MM, Mowla SJ, Tiraihi T.Nucleostemin, a coordinator of self-renewal, is expressed in rat marrow stromal cells and turns off after induction of neural differentiation.Neurosci Lett. 2005;390(2):81-6.

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Supplement2.Adenoviral vector tansfection of MSCs and Identification of DAPI labeled MSCs

A.The ability of the adenoviral vector to infect MSCs.MSCs were transfected with EGFP recombinant adenoviruses and the percentage of MSCs expressing EGFP and the percentage of MSCs expressing human SDF-1α(B) and human VEGF165(C) were analyzedUnder fluorescence microscopy.D.The obviousnuclear and faint cytoplasmic blue fluorescence. Virtually100% of the cultured cells are labeled by DAPIbefore transplantation (100×).

Supplement 3.The survivalrate of MSCs transfected with Ad-VEGF or Ad-SDF-1.

MSCs transfected with Ad-VEGF and/or Ad-SDF-1 showed the significantly decreased cell apoptosis by TUNEL assay.Red fluorescence showed cell apoptosis postive in MSCs, blue fluorescence showed DAPI counterstainingof cell nucleus in the same visual field(magnification:200×).

Supplement 4.Increased survival rates of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 in MI hearts.

A-D.Representative sections of survivingMSCs transfected with Ad-VEGF and/or Ad-SDF-1 in infarcted hearts. Blue fluorescence showed DAPI counterstainingof cell nucleus in the same visual field(magnification:200×).E.Semi-quantitative assay of survival rate of MSCsin infarcted hearts. *P < 0·05 vs. other groups,#P＞0.05 vs.Ad-VEGF-MSC group. F.Quantitative assay of survival rate of MSCsin infarcted hearts.*P < 0·05 vs. other groups;#P＞0.05 vs.Ad-VEGF-MSC group;#P< 0·05 vs. Ad-VEGF-EGFP-MSC and Ad-EGFP-MSC groups.

Supplement 5.MSCs transfected with Ad-VEGF or Ad-SDF-1differentiationtoward the endothelial cell.

VEGF or SDF-1did significantly inducedifferentiationof MSCs into endothelial cell14 days after hSDF-1 treatment.Red fluorescence showed CD31 in MSCs, blue fluorescence showed DAPI counterstainingof cell nucleus in the same visual field(magnification:200×).

Supplement 6.MSCs transfected with Ad-VEGF or Ad-SDF-1differentiationtoward the cardiocytes.

VEGF or SDF-1did not significantly inducedifferentiationof MSCs into cadiocytes 14 days after hSDF-1 treatment.Red fluorescence showed cTnt+and MHC in MSCs, blue fluorescence showed DAPI counterstainingof cell nucleus in the same visual field(magnification:200×).

Supplement 7.The change of proliferation of MSCs transfected with Ad-VEGF or Ad-SDF-1.

After MSCs transfected with Ad-VEGF or Ad-SDF-1 were cultured for three days, the effect of VEGF or Ad-SDF-1 on MSCsproliferation was evluated by MTT method. *P<0·05 vs. other groups;#P<0·05 vs. other groups.