Radiolabeling Human Peripheral Blood Stem Cells for Positron Emission Tomography (PET) Imaging in Young Rhesus Monkeys

Abstract

These studies focused on a new radiolabeling technique with copper (64Cu) and zirconium (89Zr) for positron emission tomography (PET) imaging using a CD45 antibody. Synthesis of 64Cu-CD45 and 89Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC) was assessed in vitro (viability, population doubling times, colony forming units). hPBSC viability was maintained as the dose of 64Cu-TETA-CD45 increased from 0 (92%) to 160 µCi/mL (76%, p>0.05). Radiolabeling efficiency was not significantly increased with concentrations of 64Cu-TETA-CD45 >20 µCi/mL (p>0.50). Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p<0.05). For 89Zr, there were no significant differences in viability (p>0.05), and a trend towards increased radiolabeling efficiency was noted as the dose of 89Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0–40 µCi/mL (p<0.05). A greater than 2,000 fold-increase in the level of 89Zr-Df-CD45 labeling efficiency was observed when compared to 64Cu-TETA-CD45. Similar to 64Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05) (growth, colony formation). Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×106 hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI), then 0.3 mCi of 89Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that 89Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.

Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases: An NHLBI Resource for the Gene Therapy Community

“…These studies support that the route and timing of vector administration are crucial for limited biodistribution to other anatomical sites Conducted extensive analysis of gonads after various routes of lentiviral vector administration prenatally and confirmed no evidence of germ cell gene transfer in males or with organ-targeted approaches Tarantal et al, 2005). These studies further support the importance of organ-targeting and vector design (Pacak et al, 2006) to limit biodistribution Used SIV-based lentiviral vectors (Kahl et al, 2008) to transduce young monkey CD34 + hematopoietic stem/ progenitor cells for autologous transplantation, and used nonmyeloablative conditioning regimens such as busulfan and fludarabine (Kahl et al, 2006;Tarantal et al, 2012a) Addressed the role of overexpression of transforming growth factor-b 1 during fetal lung development, using an intrapulmonary gene transfer approach , providing a new model to explore lung disease that may allow greater insight into human lung development Reported that AAV8-mediated hepatic gene transfer in infant monkeys is safe and efficient but less stable when compared with adolescent animals Wang et al, 2011) Showed that in utero delivery of an AAV2/5-human FVII vector in the late third trimester confers therapeutic expression of human FVII at birth, which was maintained above baseline levels for at least 2 months, and that readministration with capsid proteins of another serotype (AAV2/8) *1 year after fetal gene delivery resulted in a further increase in plasma levels (Binny et al, 2012) Evaluated stem and progenitor cell age-related differences (fetal through aged) and showed significant differences across the lifespan (e.g., endothelial, hematopoietic, mesenchymal) (Lee et al, 2004Hacia et al, 2008;Kim et al, 2008;Shelley et al, 2012) Developed in vivo imaging techniques (PET, BLI) (Tarantal et al, , 2009(Tarantal et al, , 2012cTarantal and Lee, 2010) that demonstrated long-term gene expression, and showed engrafted foci of human cells at sites not appreciated by the collection of blood or bone marrow Showed high levels of firefly luciferase expression with no adverse effects up through *8 years postnatal age when organ-targeted fetal gene transfer approaches were used (e.g., intrathoracic, intrapulmonary, intramyocardial, intraportal, intrahepatic; lentiviral vectors or AAV serotypes) (Fig. 2).…”

“…Novel aspects of the program include the following: a strong track record in a collaborative team approach, strengths in translational research, unique capabilities in the study of fetal/pediatric nonhuman primate models of human development and disease, and the use of novel in vivo imaging modalities including positron emission tomography (PET) and bioluminescence imaging (BLI). The development and application of new in vivo imaging technologies have provided new ways to monitor long-term gene expression and the trafficking of transplanted cells in real time (Tarantal et al, , 2009(Tarantal et al, , 2012c. The Center also highlights a state-ofthe-art fetal therapy program with many ultrasound-related techniques and procedures that have been applied comparable to those used in humans (Tarantal, 2005).…”

“…The advantages of in vivo imaging include that each individual can be followed longitudinally in real time, conferring a large statistical advantage, and animal-toanimal variability is reduced as a confounding variable. Studies have also shown that in vivo BLI is highly effective for monitoring long-term gene expression, identifying foci of engrafted cells, and assessing cell trafficking when combined with PET (Tarantal et al, 2012c).…”

Lost signature: progress and failures in in vivo tracking of implanted stem cells

“…Furthermore, 89 Zr was shown to provide a better decay profile for PET (Tarantal et al 2013). 89 Zr labeling is also an improvement about 64 Cu for PET studies, as it leads to significantly higher per-cell activities (Tarantal et al 2013). A recently researched cell labeling method for PET is radiomanganese (Ittrich et al 2013).…”

Pre- and postmortem imaging of transplanted cells

“…A properly constructed compound can identify the transplanted stem cells inside the host body. Tarantal et al, 9 showed that injection of 89 Zr conjugated to human-specific CD45 antibody into the circulatory system of Rhesus monkeys facilitated imaging of the biodistribution of grafted human stem cells with PET. Another interesting application of noninvasive PET imaging was geared at differentiation of cancer stem cells from healthy brain cells.…”