MRI detection of FePro labeled long-term cultured frozen and fresh CB AC133+ EPCs in glioma.

<p>At days 10–15 (A, B) and 25–30 (E, F) of the primary culture, cells were labeled with FePro and cryopreserved for few weeks. On the day of IV administration, the cells were thawed, incubated for 1–2 hours in stem cell media, washed and IV injected. A control group of rats received freshly prepared FePro labeled cells at 10–15 (C, D) and 25–30 (G, H) days of cultures. Seven days after cell administration multi-echo gradient-echo MRI were obtained using a 7 Tesla small animal MRI system. All animals receiving either frozen or fresh FePro labeled cells exhibited low signal intensity areas around tumors (arrows). Corresponding DAB enhanced Prussian blue stained sections showed iron positive cells at the tumor margins. Both frozen and fresh FePro labeled cells migrated and accumulated in tumor sites.</p

Effect of cryopreservation on <i>in vivo</i> angiogenic properties of CB AC133+ EPCs-immunohistology.

<p>At days 10–15 and 25–30 of the primary culture cells were labeled with FePro and cryopreserved for few weeks. Seven days after IV administration of thawed FePro labeled cells to glioma bearing rats, tissues were harvested and analyzed by DAB enhanced Prussian blue staining, FITC conjugated tomato lectin (green) and Rho conjugated antibodies that recognized vWF and CD31 expression. Panels A–C depict tissue sections of animals receiving frozen labeled cells that were cultured for 10–15 days. Control animals received 10–15 days cultured non-cryopreserved FePro labeled cells (D–F). Same experiments were done with cells expanded for 25–30 days. Tissue sections from the animals receiving frozen AC133+ are shown in panels G–I, while the section from control group receiving fresh cells are shown in J–L. Scale bar = 100 µm.</p

CB AC133+ EPCs-expression of cell surface markers during long term <i>in vitro</i> culture.

<p>The data depicts CD133 and CD34 protein expression levels in cells cultured for 5, 11 and 25 days and the levels of CD117 in cells cultured for 25 days (<b>A</b>). Flow cytometric histograms from one representative experiment are shown (n = 3). At least 10,000 live gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype controls are shown as black histograms. Panel B shows cells induced to differentiate at day 25–30 of primary culture.</p

MRI relaxation parameters in tumors.

<p>Analyses of R2* values normalized to contralateral normal hemisphere (an indirect indicator of iron positive cell accumulation ) showed significantly higher (p<0.05) accumulation of iron positive cells in animals that received previously cryopreserved, FePro labeled CB AC133+EPCs that were <i>in vitro</i> expanded for 10–15 and 25–30 days. Bars: means ± SD. * p<0.05 frozen day 10–15 versus fresh day 25–30; <b>^§</b> p<0.05 frozed day 25–30 versus fresh day 10–15 and fresh day 25–30.</p

CB AC133+ EPCs expression of CD31, vWF and KDR and DiI-Ac-LDL uptake in differentiated progenitors – effect of FePro labeling and cryopreservation.

<p>Expression of CD31, VEGFR2 and vWF in differentiated cells that were prior to differentiating (at days 25–30 of the primary culture) labeled with FePro and cryopreserved for few weeks (D, E, F and G). Control cells were induced to differentiate at days 25–30 of the primary culture without previous FePro labeling and cryopreservation (A, B and C). Positive signals for CD31, VEGFR2 and vWF were visualized with a FITC conjugated secondary antibody (green). Nuclei were visualized with DAPI (blue). VEGFR2 positive (middle panels B and E) cells also exhibited the uptake of DiI-Ac-LDL (red). Representative photomicrographs (40×) of differentiated cells. Scale bar = 100 µm.</p