Optimized longitudinal monitoring of stem cell grafts in mouse brain using a novel bioluminescent/near infrared fluorescent fusion reporter.

Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achievedin 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 human bone marrow derived 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 and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were characterized 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 starting 1 week after cell injection when the blood brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luciferase substrate performances i.e. D-luciferin (D-Luc) (25μM/kg or 943μM/kg) or CycLuc1 (25μM/kg).

Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential towards adipocytes, chondrocytes and osteocytes suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1x10(5) cells using both fluorescence and bioluminescence imaging. The highest bioluminescent signals (~1x10(7) ph/s) were achieved 15 minutes after injection of D-luciferin (943μM/kg). This allowed us to monitor as low as 1x10(5) hMSCs for the subsequent 7 weeks without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.