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Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter
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Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter
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Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter
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Journal Article
Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter
2017
Overview
Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood–brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 μM/kg or 943 μM/kg) or CycLuc1 (25 μM/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 × 105 cells using both fluorescence and BLI. The highest bioluminescent signals (∼1 × 107 photons per second) were achieved 15 min after the injection of D-Luc (943 μM/kg). This allowed us to monitor as low as 1 × 105 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.
Publisher
SAGE Publications,Sage Publications Ltd,SAGE Publishing
