MRI tracking of FePro labeled fresh and cryopreserved long term in vitro expanded human cord blood AC133+ endothelial progenitor cells in rat glioma

Abstract

Background: Endothelial progenitors cells (EPCs) are important for the development of cell therapies for various diseases. However, the major obstacles in developing such therapies are low quantities of EPCs that can be generated from the patient and the lack of adequate non-invasive imaging approach for in vivo monitoring of transplanted cells. The objective of this project was to determine the ability of cord blood (CB) AC133+ EPCs to differentiate, in vitro and in vivo, toward mature endothelial cells (ECs) after long term in vitro expansion and cryopreservation and to use magnetic resonance imaging (MRI) to assess the in vivo migratory potential of ex vivo expanded and cryopreserved CB AC133+ EPCs in an orthotopic glioma rat model.

Materials, methods and results: The primary CB AC133+ EPC culture contained mainly EPCs and long term in vitro conditions facilitated the maintenance of these cells in a state of commitment toward endothelial lineage. At days 15-20 and 25-30 of the primary culture, the cells were labeled with FePro and cryopreserved for a few weeks. Cryopreserved cells were thawed and in vitro differentiated or i.v. administered to glioma bearing rats. Different groups of rats also received long-term cultured, magnetically labeled fresh EPCs and both groups of animals underwent MRI 7 days after i.v. administration of EPCs. Fluorescent microscopy showed that in vitro differentiation of EPCs was not affected by FePro labeling and cryopreservation. MRI analysis demonstrated that in vivo accumulation of previously cryopreserved transplanted cells resulted in significantly higher R2 and R2* values indicating a higher rate of migration and incorporation into tumor neovascularization of previously cryopreserved CB AC133+ EPCs to glioma sites, compared to non-cryopreserved cells.

Conclusion: Magnetically labeled CB EPCs can be in vitro expanded and cryopreserved for future use as MRI probes for monitoring the migration and incorporation to the sites of neovascularization.

Figure 1. CB AC133+ EPCs-expression of cell surface markers during long term in vitro culture.

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 (A). 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.

Figure 2. CB AC133+ EPCs expression of CD31, vWF and KDR and DiI-Ac-LDL uptake in differentiated progenitors – effect of FePro labeling and cryopreservation.

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.

Figure 3. MRI detection of FePro labeled long-term cultured frozen and fresh CB AC133+ EPCs in glioma.

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.

Figure 4. MRI relaxation parameters in tumors.

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 in vitro expanded for 10–15 and 25–30 days. Bars: means ± SD. * p<0.05 frozen day 10–15 versus fresh day 25–30; ^§ p<0.05 frozed day 25–30 versus fresh day 10–15 and fresh day 25–30.

Figure 5. Effect of cryopreservation on in vivo angiogenic properties of CB AC133+ EPCs-immunohistology.

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.