Characterization of mesenchymal stem cells and <i>CTNS</i>(−/−) mutant target cells.

<p>(a) FACS analysis (left) of bmMSC surface markers (blue) and isotype controls (red) and bmMSC differentiation from baseline (upper right) to Alizarin Red S-stained calcium-containing osteogenic (middle right) and Oil-Red O-stained adipocyte (bottom right) phenotypes. (b) same FACS and differentiation analysis of amMSC cells. (c) schematic diagram depicting the common 57 Kb deletion removing the 5′ portion of the <i>CTNS</i> gene and PCR-proven genotype of control and mutant fibroblasts from a cystinosis patient. Intracellular cystine content of control and homozygous <i>CTNS</i> mutant fibroblasts is shown on the right bar-graph.</p

Analysis of stem cell microvesicles and their effects on cystine content of <i>CTNS</i>(−/−) mutant fibroblasts.

<p>(a) diameter of bmMSC microparticles released into conditioned medium. (b) effect of increasing amounts of bmMSC microvesicles on cystine in mutant fibroblasts after 24 hour incubation, compared to the effect of 1 mM cysteamine. (c) diameter of amMSC microparticles released into conditioned medium. (d) effect of increasing amounts of amMSC microvesicles on cystine in mutant fibroblasts after 24 hour incubation, compared to the effect of 1 mM cysteamine and in the presence of annexin V. Statistical significance is indicated by the corresponding p-value.</p

Effect of co-culture with CTNS-expressing cells on cystine content of mutant <i>CTNS</i> cells.

<p>(a) Cystine content of <i>CTNS</i>(−/−) fibroblasts co-cultured with increasing amounts of wildtype amMSC. (b) Cystine content of GFP-tagged <i>CTNS</i>(−/−) ciPTEC isolated by FACS after co-culture with unlabelled wildtype amMSC, mutant ciPTEC or <i>CTNS</i>-corrected ciPTEC as donors. Statistical significance is indicated by the corresponding p-value.</p

amMSC microvesicle transfer of CTNS^Red to acidic intracellular compartment of <i>CTNS</i>(−/−) mutant fibroblasts.

<p>(a) amMSC stably transfected with CTNS^Red immunofluorescent protein. (b) CTNS^Red in amMSC microvesicles. (c) single confocal plane showing CTNS^Red protein in amMSC (circles) and in co-cultured GFP-tagged <i>CTNS</i>(−/−) mutant fibroblasts (arrows). Dashed line indicates amMSC cluster also shown in brightfield insert. (d) fusion of CTNS^Red particle with LysoTracker stained endosome/lysosome (arrowhead), shown in insert at higher magnification.</p