In Vivo Tracking Of Stem Cells Transplanted In Spinal Cords With Radionuclide Imaging

OBJECTIVES Stem cell transplantation as a promising treatment for many disorders categorized incurable previously is attracting lots of interest recently. In spite its great therapeutic potentials, safety and long term biological outcome of the implanted stem cells inside host's body are the issues of concerns before it can be pushed on into clinical trial. In animal experiments, tissues can be sampled at anytime to monitor the existence and/or transformation of the implanted cells. But such an invasive assessment is definitely out of the question in patients. It is thus fairly critical for the researchers to find a non-invasive way to verify whether the cells injected are alive, where they migrate to and what kinds of cell they finally differentiate into. With progress in hardware, software and radiochemistry, nuclear medicine imaging using radiolabeled agents specifically binding to the markers of implanted cells, co-varying with their differential states, could be feasible to monitor the entire life-span of the implanted cells. The first problem to be solved is whether the tiny number of stem or progenitor cells grafted in vivo could be detected with radionuclide imaging techniques. With this in mind, two markers of stem or progenitor cells were selected as imaging targets and radionulcide imaging was performed to track the cells transplanted in animal spinal cords. MARKER 1-TRANSFERRIN RECEPTORSMETHODS Isolated from human fetal blood and expanded with in vitro culture, human mesenchymal stem cells (hMSCs) were transplanted into rabbit spinal cord. Transferrin receptor expression of hMSCs was examined with flow cytometry, cell and tissue immunofluorescence. Fe satured human transferrin was labeled with 125I and 131I via a modified lodogen procedure. Radioligand binding assay was performed to quantify the transferrin receptors expression. The 2nd and 10thday after transplantation, radioiodinated transferrin was injected into subarachnoid space and a series of gammar camera images with a pinhole collimator were acquired. As imaging control, rabbits were transplanted with same volume of PBS or 13II labeled human serum albumin was injected as tracer. Autoradiography was carried out to confirm results of in vivo imaging.RESULTS Transferrin receptors expression of hMSCs in culture and in vivo 2 days after transplantation was demonstrated with flow cytometry, immunofluorescent staining. Radioligand binding assay showed that the expressing level was high (about 1.1 x 105 per cell), and radioiodinated transferrin could bind to hMSCs with high affinity (Kd=0.982 nM). On 2nd day after transplantation, accumulation of radioactivity at the hMSCs injection site was detected with scintigraphy at 16hr~24hr post-injection of 131I-transferrin radiotracer into subarachnoid space. The control experiments with 131I labeled human serum albumin as tracer and PBS as grafts as well as ex vivo and in vitro autoradiography demonstrated the specificity of the implanted hMSCs' uptake of radiotracer. However, on the 10th day of transplantation, negative imaging was observed at hMSCs injection site on scintigraphic images, and non-expression of transferrin receptors on implanted cells was confirmed by immunofluorescent staining and autoradiography. MARKER 2-DOPAMINE D2 RECEPTORSMETHODS Founded by transduction of telomerase gene to neurospheres separated from the subventricular zone of natural aborted fetus brain, neural progenitor cells (hNPC-TERT) were grafted into rabbit spinal cord. Dopamine D2 receptor expression on hNPC-TERT cell was examined with reversal transcription PCR (RT-PCR), cell and tissue immunofluorescence. Radioligand binding assaywas performed to quantify the D2 receptor expression. aC-Raclopride was synthesized with a column chromatography method. On the 2nd day of cells transplantation, nC-Raclopride was injected intravenously, and living animals or ex vivo spinal cords accepted PET scans. A control study was performed on animals implanted with HeLa cells.RESULTS RT-PCR showed the dopamine D2 receptors was p...