Fig. 4

Mitochondrial morphology and dynamics in PAM-MSCs and MAT-MSCs. A Imaging of functional mitochondria in PAM-MSCs and MAT-MSCs. MSCs were incubated with TMRE and analyzed by confocal microscopy as detailed in Materials and Methods. Addition of the uncoupler FCCP vanished completely the fluorescent signal (indicated in the right upper corner of the main images). Digital enlargements of details are shown at the bottom of the pictures after false color rendering and thresholding to remove background; ImageJ tools were used with identical parameter for both PAM- and MAT-MSCs. The images shown are representative of three analyses with as many biological replicates yielding similar results. The histograms on the right show statistical morphometric analysis of the mitochondrial compartment assessed by ImageJ tools. Ten–fifteen cells/optical field from 4–5 fields of each biological replicate were randomly chosen, and the following parameters measured treating the mitochondria as objects: (1) averaged normalized TMRE pixel density/cell; (2) elongation/cell, defined as (mean perimeter)²/(4π × area) of all the mitochondria per cell; averaged Feret’s diameters, defined as the longest distance between any two points along the selection boundary of an object; interconnectivity/cell, defined as the (mean area)/(mean perimeter) ratio. *, P < 0.05; **, P < 0.005. B Representative immunoblots of mitochondrial markers. CxII SDHB, subunit B of the succinate dehydrogenase—complex II or the respiratory chain; TOMM40 and TOMM20, subunits of the translocator complex of the outer mitochondrial membrane. The histogram on the right shows the averaged normalized densitometric analysis ± S.E.M. of four independent MSCs preparations. C Representative immunoblots of mitochondrial dynamics markers. MFN1 and MFN2, mitofusins 1 and 2; OPA1, optic atrophy protein 1; DRP1, dynamin-related protein 1; LC3A/B-I, unmodified light chain 3A/B; LC3A/B-II, PE-conjugated light chain 3A/B. The histogram on the right shows the averaged normalized densitometric analysis ± S.E.M. of three independent MSCs preparations