TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF).

One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.

Noninvasive discrimination of human normal cells and malignant tumor cells by phase-shifting laser microscopy.

The noninvasive discrimination of cancer cells from normal cells in adherent culture by the measuring of the phase shift using phase-shifting laser microscopy (PLM) was investigated with the aim of noninvasive quality control of cell processing for transplantation. A human prostatic carcinoma epithelial cell line (PC-3) and human hepatocellular carcinoma cell lines (Hep3B, PLC, HLF, and Huh7) showed markedly lower phase shifts as measured by PLM than those of human prostate epithelial cells (PREC) and cryopreserved human hepatocytes, respectively, although there was no apparent difference in cell morphology between these sets of cells. Mixed cultures of PC-3 and PREC, in which the percentages of PC-3 cells were determined to be 43.3%, 14.0%, and 10.6% by immunofluorescence staining, were prepared, and the percentages were calculated to be 50.0%, 8.0%, and 2.5% using the distribution of the phase shift data of the cells, respectively. In conclusion, these adherent cancer cells can be noninvasively discriminated from normal adherent cells by phase shift measurement using PLM, and the sensitivity of the method of detecting cancer cell contamination reached 10%.

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy.

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4',6-diamidine-2'-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G(2)/M phase was markedly higher than that of cells in the G(0)/G(1) phase. Even in the synchronous cultures to G(0)/G(1) and G(2)/M cell cycle phases, the laser phase shift of the cells in the G(2)/M phase was markedly higher than that of the cells in the G(0)/G(1) phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.