In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation
Figure 5
mAT-MASCeGFP-DiD distribution and characterization. (a, b, c) Real-time PCR analysis was performed to detect transgenic cells from recipient mice. Calibration curves, respectively, for the gene eGFP (a) and beta-actin (b) were produced to extrapolate the absolute concentration of both genes for each analyzed tissue. In (c) the relative amount of eGFP cells per 105 nuclei detected in lung, spleen, liver, injected tibialis anterior muscle (TA Inj.), adipose tissue and skin near the site of injection (ADIP. Inj., and SKIN Inj.), diaphragm, heart, contralateral adipose tissue (ADIP. Contral.), and tibialis anterior muscle were reported. eGFP column indicated a representative value obtained from eGFP transgenic tissue. mAT-MASCeGFP presence 7 days after cell delivery was confirmed by immunofluorescence assays (d, e), green fluorescence (calibration bar 20 μm) (j, l) red fluorescence, calibration bar 20 (μm). Lambda scan analysis was performed to confirm the specificity of the signal. Emission spectra for DiD (g) (red line) and eGFP, Alexa 555 (i) (red line) were singularly analyzed and appeared distinct from the tissue autofluorescence emission spectra (g, i) gray lines. (k) Cells exhibiting at the same time spectra specific for DiD emission and eGFP (Alexa 555 emission) were found at the injected site (red line) and not in tissue sections obtained from not-injected mice (grey lines). Detection of male mAT-MASCeGFP by FISH analysis (m, n, o). The presence of Y-chromosome positive cells (red spots) in female tissues suggested that viable donor cells persisted in tibialis anterior muscle (calibration bar 50 μm). (d, e, f) By costaining cells for eGFP (green fluorescence) and MCM5 (red fluorescence) double positive cells were detected, indicating that injected cells were viable and proliferated.
