Cellular Bioenergetics Is an Important Determinant of the Molecular Imaging Signal Derived From Luciferase and the Sodium-Iodide Symporter

Abstract

Rationale Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number can influence the molecular imaging signal obtained from reporter genes. Objective The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase(fluc) and human Na-I symporter(hNIS) labeling of cardiosphere-derived cells (CDCs) was investigated. Methods and Results 18FDG uptake, ATP levels, 99mTc-pertechnetate uptake and bioluminescence were measured in vitro, in adherent and suspended CDCs. In vivo dual isotope SPECT-CT imaging or bioluminescence imaging (BLI) were performed 1hr and 24hrs following CDC transplantation. SPECT quantification was performed using a phantom for signal calibration. Cell loss between 1hr & 24hrs post-transplantation was quantified by qPCR and ex vivo luciferase assay. Cell dissociation followed by suspension for 1hr resulted in decreased glucose uptake, cellular ATP, 99mTc uptake and BLI signal by 82%, 43%, 42%, and 44% respectively, when compared to adherent cells, in vitro. In vivo 99mTc uptake was significantly lower at 1hr, when compared to 24hrs following cell transplantation in the non-infarct (p<0.001, n=3) and infarct (p<0.001, n =4) model, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1hr than at 24hrs (p<0.01), with the BLI signal being higher when CDCs were suspended in glucose-containing medium compared to saline(PBS). Conclusion Adhesion is an important determinant of cellular bioenergetics, 99mTc-pertechnetate uptake and BLI signal. BLI and NIS imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.

The Sodium/Iodide Symporter (NIS): Molecular Physiology and Preclinical and Clinical Applications

“…Chang and colleagues (162) transduced NIS into cardiosphere-derived cells (CDCs) to determine how different methods of cell separation prior to injection would affect cell metabolism and bioenergetics and ultimately the efficiency of the engraftment of the cells. Their results may eventually help optimize the preparation conditions for CDC transplantation.…”

Hyaluronic acid-serum hydrogels rapidly restore metabolism of encapsulated stem cells and promote engraftment

“…We have previously demonstrated that cell dissociation and suspension rapidly down regulate glucose uptake, metabolism and ATP levels[1]; suspension also predisposes cells to anoikis[25, 26]. Stem cells utilize glucose as their main energy source[27].…”

“…The cDNA encoding the hNIS (human sodium iodide symporter) gene or the cDNA pGL4.10[luc2] encoding firefly luciferase (Promega) was sub-cloned in place of eGFP into the vector RRLsin18.cPPT.CMV.eGFP.Wpre, resulting in plasmids designated cpPPT.CMV.hNIS or pPPT.CMV.fLuc as previously described[1]. Viral vectors were produced and titered as described previously[1]. For genetic labeling, rat CDCs were transduced at a multiplicity of infection (MOI) of 20, yielding transduction efficiencies of >70% for hNIS expression and >90% for fLuc expression.…”

“…Seahorse Bioscience XF instrument (XF24 with Islet capture plates)[1, 21] was used to measure oxygen consumption rate (OCR). Respiratory rates were measured as basal rates.…”

“…An important obstacle to sustained functional benefits is very low levels of acute stem cell retention and engraftment. We have previously demonstrated[1] using cardiosphere-derived cells (CDCs)[2] that dissociation leads to rapid depression of stem cell bioenergetics, indicating a relationship between cellular metabolism and adhesion. Molecular imaging studies using micro-PET/CT of ex vivo 18 FDG-labeled CDCs reveal that ~80% of injected CDCs are lost into the lungs and systemic circulation in the first hour following intra-myocardial transplantation[3].…”