HLH-30/TFEB Is a Master Regulator of Reproductive Quiescence.

All animals have evolved the ability to survive nutrient deprivation, and nutrient signaling pathways are conserved modulators of health and disease. In C. elegans, late-larval starvation provokes the adult reproductive diapause (ARD), a long-lived quiescent state that enables survival for months without food, yet underlying molecular mechanisms remain unknown. Here, we show that ARD is distinct from other forms of diapause, showing little requirement for canonical longevity pathways, autophagy, and fat metabolism. Instead it requires the HLH-30/TFEB transcription factor to promote the morphological and physiological remodeling involved in ARD entry, survival, and recovery, suggesting that HLH-30 is a master regulator of reproductive quiescence. HLH-30 transcriptome and genetic analyses reveal that Max-like HLH factors, AMP-kinase, mTOR, protein synthesis, and mitochondrial fusion are target processes that promote ARD longevity. ARD thus rewires metabolism to ensure long-term survival and may illuminate similar mechanisms acting in stem cell quiescence and long-term fasting.

Initial graft size and not the innate immune response limit survival of engrafted neural stem cells.

Transplantation of neural stem cells (NSCs) appears to be a promising regenerative therapy for a variety of neurological disorders. Nevertheless, NSC engraftment is limited by the number of surviving cells. To maximize stem cell-mediated effects, timing of implantation and cell number have to be precisely evaluated. Here, a transgenic murine NSC line was optimized for high expression levels of the imaging reporters Luc2 and copGFP. NSCs of 150 000, 75 000, 15 000 or 1500 cells or Hanks buffered salt solution were implanted into the striatum of nude mice. The survival of NSCs was monitored with in vivo bioluminescence imaging (BLI) over 2 weeks and brain sections were histologically analysed for glial cells of the innate immune system. The longitudinal in vivo BLI data revealed a significantly reduced viability with the highest rate for 150 000 engrafted NSCs. The cell loss was not correlated with the number of Iba-1+ immune cells nor GFAP+ astrocytes. Histological quantification of copGFP+ cells at 14 days postimplantation confirmed the in vivo data with the highest density of copGFP+ cells in the 150 000-cell graft and the highest survival rate for 1500 cells/graft. In conclusion, regenerative therapies should strictly evaluate the maximal number of stem cells to be transplanted in one location, as the results suggest that there is a critical limit of cells able to survive in the adult brain. Survival is limited by availability of oxygen and nutrients but not the inflammatory response induced by the implantation.

Novel bimodal iron oxide particles for efficient tracking of human neural stem cells in vivo.

AIMS: We validated novel bimodal iron oxide particles as substitute of ferumoxides for efficient labeling of human neural stem cells (NSCs). The dextrane-coated FeraTrack Direct (FTD)-Vio particles have additional far-red fluorophores for microscopic cell analysis. METHODS: MR relaxometry, spectrophotometric iron determination and microscopy are used for characterization in vitro and in vivo. RESULTS: Efficient uptake is not transfection agent-dependent. FTD-Vio594 labeling had no influence on viability, proliferation, migration and differentiation capacity. It allows MRI-based tracking of engrafted NSCs in mouse brain up to 11 days, complemented by bioluminescence imaging of firefly luciferase expressed by the engrafted cells. CONCLUSION: Our results highlight the FTD-Vio594 particles as safe and sensitive substitute of ferumoxides for longitudinal tracking of NSCs in preclinical studies.

Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks.

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.

Proof of concept of an automatic tool for bioluminescence imaging data analysis.

Bioluminescence Imaging (BLI) is an important molecular imaging tool to assess complex biological processes in vivo. BLI is a sensitive technique, which is frequently used in small-animal preclinical research, mainly in oncology and neurology. Tracking of labeled cells is one of the major applications. However, BLI data analysis for the segmentation of up-taking regions and their quantification is not trivial and it is usually an operator-dependent activity. In this work, a proof of concept of an automatic method to analyze BL images is presented which is based on a multi-step approach. Different segmentation algorithms (K-means, Gaussian Mixture Model (GMM), and GMM initialized by K-means) were evaluated and an adequate image normalization step was suggested to include the background bioluminescence in the data analysis process. K-means segmentation is the most stable and accurate approach for different levels of signal intensity.