Radiolabeling and In Vivo Imaging of Transplanted Renal Lineages Differentiated from Human Embryonic Stem Cells in Fetal Rhesus Monkeys

Abstract

Purpose The goals of this study were to optimize radiolabeling of renal lineages differentiated from human embryonic stem (hES) cells and use noninvasive imaging (positron emission tomography (PET) and bioluminescence imaging (BLI)) to detect the cells in fetal monkeys post-transplant. Procedures hES cells expressing firefly luciferase (5×106) were radiolabeled with the optimized concentration of 10 μCi/ml 64Cu-PTSM then transplanted under ultrasound guidance into early second trimester fetal monkey kidneys. Fetuses were imaged in utero with PET and tissues collected for analysis 3 days post-transplant. Fetal kidneys were imaged ex vivo (PET and BLI) post-tissue harvest, and serial kidney sections were assessed by PCR for human-specific DNA sequences, fluorescent in situ hybridization (FISH) for human-specific centromere probes, and immunohistochemistry (IHC) to assess engrafted cells. Results Transplanted cells were readily imaged in vivo and identified at the site of injection; tissue analyses confirmed the imaging findings. Using a semi-quantitative method, one in approximately 650 cells in the kidney was shown to be of human origin by PCR and FISH. Conclusions These studies suggest that hES cells differentiated toward renal lineages can be effectively radiolabeled, transplanted into fetal monkey kidneys under ultrasound guidance, monitored with PET post-transplant, and identified by PET, BLI, PCR, FISH, and IHC post-tissue harvest.

Lysophosphatidic Acid Protects Human Mesenchymal Stromal Cells from Differentiation-Dependent Vulnerability to Apoptosis

“…MSC were transduced to express firefly luciferase (MSC-Luc) for in vivo studies as previously described. 25,26 Cells were cultured under standard conditions in a humidified incubator and utilized at passages 5-6. To induce osteogenic differentiation, cells were cultured in either osteogenic media (OM: GM supplemented with 10 mM b-glycerophosphate and 50 mg/mL ascorbate-2-phosphate; Sigma-Aldrich, St.…”

“…25,26 Briefly, mice were injected with D-Luciferin, Firefly (Caliper, Hopkinton, MA; 10 mL/g body weight), and luminescence was measured using Living Image software (Perkin Elmer). Total photons per second per centimeter were recorded from each bioluminescent region of interest.…”

Regeneration of the Kidney - Viewed from ES Cell

“…There is a report indicating that administration of MSC to a rat model of ischemia-reperfusion-induced acute renal failure improved renal function, whereas administration of syngeneic fibroblasts did not. MSC expressed more growth factors VEGF, HGF and IGF-1 than fibroblasts ( Togel F., et al (2005)). Some of these factors are known to modulate kidney function or repair.…”

“…This may be due to the various cytokines secreted from transplanted MSC that organize an appropriate microenvironment for the kidney repair in autocrine and paracrine fashion. To be sure, MSC secretes various cytokines such as VEGF, HGF and IGF-1 (Togel F.,et al (2005).). Injection of bone marrow-derived MSC into peritoneal cavity attenuated the cisplatin-induced renal failure without the engraftment of MSC in the kidney.…”

Technology Advancement for Integrative Stem Cell Analyses

“…112 With the help of molecular imaging techniques that enable the longitudinal, noninvasive assessment of the in vivo behavior of stem cells, the studies of cell survival, distribution, immunogenicity, and tumorgenicity have been actively performed after stem cell transplantation. With advances of labeling techniques, many groups have demonstrated ''Direct labeling techniques'' such as iron particle label, 122,123 radionuclide label 124,125 for noninvasive imaging of stem cell migrations, engraftment, and morphological differentiation. In addition, the reporter gene labeling has been shown, coupling a reporter gene with a complimentary reporter probe where it is …”