Figure 3.

Sca-1+/CD31− CSCs^hTERT exhibit stem cell potency. For phenotypic characterization, Sca-1+/CD31− CSCs^hTERT expressing GFP (green) were analyzed by immunostaining (A) and flow cytometry (B) with different cell surface antibodies (red); (C) real-time PCR was performed to detect both endogenous mTERT and transduced hTERT transcripts in primary Sca-1+ CSCs and immortalized Sca-1+/CD31− CSCs^hTERT. Data represent mean ± SD from three independent experiments (* p < 0.05; ** p < 0.01). ND, not determined; (D) for proliferation analysis, Sca-1+/CD31− CSCs^hTERT were seeded at 1 × 10³ cells/well in 96-well microplates, cultured with Mesencult Basal Medium supplemented with cytokines for six days, and analyzed by WST-1 assay. Data represent mean ± SD from four independent experiments; and (E) Sca-1+/CD31− CSCs^hTERT exhibit multi-differentiation potential. Differentiation was analyzed by immunostaining with a cardiomyocyte marker (cTnT, red), an endothelial marker (vWF, red), and by Oil-Red O staining (red) or Alizarin Red S staining (red). Nuclei were stained with DAPI (blue). Scale bars = 20 μm.