In Vivo Imaging to Monitor Trafficking and Engraftment of Human CD34+ Hematopoietic Stem and Progenitor Cells in Rhesus Monkeys

Significant advancements have been made in the use of noninvasive imaging techniques which includes dramatic improvements in resolution and sensitivity, and imaging systems designed specifically for animals. The non-invasive nature of these techniques allows longitudinal assessments over time, and the evaluation of protocols that could be applied in a human clinical setting. Imaging is also of great utility in situations when it is unclear where to focus the analyses, such as the challenges presented by systemic administration of transplanted cells, which can traffic to different anatomical sites. Our prior studies have shown that we can monitor gene expression in fetal and infant monkeys using reporter genes, microPET, and optical imaging. We have also shown that human mobilized peripheral blood stem cells (hPBSC) from adult male donors and male cord blood CD34+ hematopoietic stem cells (HSC) can readily engraft in fetal rhesus monkeys and persist over time. Other recent findings suggest that growth factor expansion of cord blood CD34+ HSC in vitro prior to transplant substantially increases the level of engraftment, with evidence of human hematopoietic cells in the rhesus host at levels significantly greater than the approximate 2% we previously reported. Our studies have also focused on new ways to use optical imaging to monitor transplanted hPBSC, cord blood HSC, and CD34+ precursors differentiated from human embryonic stem cells (hESC) that express firefly luciferase and a drug resistance gene, and the development of PET techniques for short-term tracking of transplanted cells. Transduced cells (1×10⁴–20×10⁶ cells/fetus) were transplanted intraperitoneally into fetal monkeys in the late first trimester under ultrasound guidance (N=27), then animals were monitored sonographically during gestation, and delivered by cesarean-section at term. At 1 week postnatal age and monthly thereafter animals were imaged for firefly luciferase expression (bioluminescence) following the intravenous injection of 100 mg/kg D-luciferin, and after the collection of blood and bone marrow for analysis. While evidence of the human Y chromosome (hSRY, hTSPY) was found in peripheral blood mononuclear cells and CD34+ immunoselected cells from marrow by qRT-PCR, the range of copies detected over time did not reflect the imaging findings. Foci of engrafted human cells were found to persist for ≥1 year, to date, in the rhesus host and in anatomical areas not previously identified (e.g., ribs, sternum, liver). In addition, ⁶⁴Cu-PTSM radiolabeled hPBSC from the same donors were injected postnatally and cell trafficking monitored without evidence of toxicity or adverse effects. These in vivo imaging studies have provided unique insights into the fate of CD34+ cells post-transplantation, and indicate the importance of these techniques for cell transplant protocols.