Radiolabeling of CD34+ stem cells for PET and gamma imaging of cell trafficking

Abstract

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Objectives: To develop and evaluate PET and gamma imaging techniques for imaging the trafficking of transplanted hematopoietic stem cells.

Methods: Expanded human CD34+ stem cells from G-CSF-mobilized peripheral blood were labeled using commercial In 111 Oxyquinoline Solution (36 µCi/27 million cells), or a similarly formulated ⁶⁴Cu 8-hydroxyquinoline solution (52 µCi/27 million cells). For labeling, 4 mL buffered saline suspensions of cells and radionuclide were incubated at 37°C for 20 m. Culture media (5 mL) was then added and incubation continued an additional 5 m. The cells were pelleted by centrifugation and radioactivity in the supernatant vs. pellet quantified to define radiolabeling efficiency. The ⁶⁴Cu- and ¹¹¹In- cell preparations were each mixed with an equal number of unlabeled cells in media (final concentration: 56 million cells/0.66mL). A control cell preparation was subjected to the same handling conditions, omitting addition of the 8-hydroxyquinoline solution of radionuclide. A 100,000 cell aliquot of each preparation was cultured for analysis of cell viability. The remaining cells were administered intravenously to NOD/SCID mice (18.7 million cells/mouse, n = 3 for each preparation). The mice were imaged at 0.5-2 h and ~20 h post-injection to assess trafficking, and sacrificed 8-weeks following the transplant to assess engraftment.

Results: Using ⁶⁴Cu-oxine and ¹¹¹In-oxine, the CD34+ stem cells were labeled with 83% and 59% efficiency, respectively. In vitro, cell viability by propidium iodide staining at 3 days post-labeling was 92%, 88%, and 75% for control expanded cells, ¹¹¹In-labeled cells, and ⁶⁴Cu-labeled cells, respectively. IndyPET images obtained with ⁶⁴Cu showed the cells to be localized in the lungs shortly post-injection, but largely in liver and spleen the following day. Planar gamma images of the mice receiving the ¹¹¹In-cells showed a similar distribution pattern. At 8 weeks post-transplantation, human cells were found in the bone marrow and spleen. Engraftment of human cells in the bone marrow was 29% ± 29 (range 0-50%; n = 3), 74 ± 18% (range 53-88%; n = 3), and 44 ± 23% (range 19-64%; n =3), respectively, for the control-expanded, ¹¹¹In-labeled, and ⁶⁴Cu-labeled cell preparations. Multi-lineage engraftment occurred in transplanted mice and consisted of human B cells (CD19+), myeloid cells (CD33+), and progenitor cells (CD34+). In addition, clonogenic progenitor cells were present in the bone marrow of engrafted mice. Human cells were also present in the spleen in the majority of the mice.

Conclusions: CD34+ stem cells can be radiolabeled with sufficient ⁶⁴Cu and ¹¹¹In to allow imaging for assessment of cell trafficking, while still retaining cell viability

Research Support (if any): Copper-64 production was supported by NCI Grant R24-CA86307.