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 achieved in the last decade using optical imaging

The in vivo roles of Sirtuin 1 and 3 in age and obesity-related decline in female fertility

Sirtuin 1 (Sirt1) and Sirtuin 3 (Sirt3), are members of the sirtuin family of NAD+-dependent deacetylases. Sirt1 exerts its function in the nucleus, whereas Sirt3 exerts its function in mitochondria. Both Sirt1 and Sirt3 are indispensable for mitochondrial function and antioxidant defences in somatic cells. Mitochondria and oxidative stress are pivotal determinants of oocyte developmental competence (also known as quality). Thus far, analyses of Sirt1 and Sirt3 activities in oocytes have been limited to in vitro knockdown and chemical inhibitor approaches. Significantly, the in vivo roles of Sirt1 and Sirt3 for female fertility have not been formally tested. Since results with whole-animal knockouts would be confounded by the indispensable requirement for Sirt1 in somatic cells, I used the Zp3-Cre-LoxP system to specifically delete Sirt1’s catalytic domain in vivo in growing oocytes (OoSirt1∆Ex4/∆Ex4). To investigate the role of Sirt3 for female fertility, I employed a whole-animal Sirt3-knockout (Sirt3-/-) mouse model.I evaluated female reproductive capacity by quantifying ovarian follicles (ovarian sections); in vitro meiotic maturation, spindle assembly and chromosome segregation (time-lapse confocal microscopy); in vitro preimplantation embryo development; expression of SIRT1 and SIRT3 transcription targets (qRT-PCR); oocyte oxidative stress and mitochondrial content (confocal imaging), and; reproductive capacity (mating trials).Surprisingly, I found that all these parameters were indistinguishable in young (2-month-old) OoSirt1∆Ex4/∆Ex4 and Sirt3-/- females compared to corresponding wild-type females. I reasoned that the roles of Sirt1 deacetylase activity in oocytes (oocyte-Sirt1) and Sirt3 in mitochondrial function and antioxidant defences might become apparent under stressed conditions. Therefore, I undertook further analyses under two in vivo stressed states: natural ageing (10-12-months) and obesity induced by high-fat diet (HFD).Very interestingly, my analysis revealed an age-accelerated decline in reproductive performance in OoSirt1∆Ex4/∆Ex4 females, which was due to a decline in oocyte quality. Interestingly, compromised oocyte quality does not impact in vivo oocyte maturation or fertilisation but leads to increased oxidative stress in preimplantation embryos inhibiting cleavage divisions. Remarkably, HFD-induced obese OoSirt1∆Ex4/∆Ex4 females showed no defects in any of the above parameters (including litter sizes) when compared to wild-type females. Also, Sirt3-/- females exhibit no defects in oocyte quality and reproductive performance even following HFD-induced obesity and natural ageing.In my PhD project, I thoroughly interrogate the in vivo roles of oocyte-Sirt1 and Sirt3 for female fertility for the first time. My data suggest that defects in aged females lacking oocyte-Sirt1 arise due to concurrent age-related changes such as reduced NAD+ and sirtuin expression levels, which may compromise compensatory mechanisms that can cover for Sirt1 loss in younger oocytes. In contrast to evidence that increasing Sirt1 activity delays ageing, my data provide some of the only in vivo evidence that loss of Sirt1 induces premature ageing. Unexpectedly, in stark contrast to the importance of Sirt3 for countering stressed states in the somatic compartment, my results reveal that Sirt3 is completely dispensable for female fertility regardless of stress-status. This might be because ATP levels are sustained in vivo through increased mitochondrial mass possibly secondary to compensatory upregulation of another sirtuin, Sirt1, which has overlapping functions with Sirt3

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 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