間葉系幹細胞の分化制御 : 三次元細胞密度変化による遺伝子発現プロファイル変化とCRISPR-Cas9システムを用いる遺伝子発現制御

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 菅野 純夫, 東京大学教授 古川 洋一, 東京大学客員教授 本田 真也, 東京大学教授 佐藤 守俊, 東京大学准教授 合山 進University of Tokyo(東京大学

Monitoring Molecular Properties of a Fluorescence Light-Up Aptamer Using Fluorescence Cross-Correlation Spectroscopy

Fluorescence light-up aptamers (FLAPs) are tools for RNA imaging, wherein the RNA of interest is appended with a FLAP sequence that can bind to a corresponding small-molecule fluorogen and enhance its fluorescence. The fluorescence properties of FLAPs have mostly been analyzed in bulk and described as the average of a large number of RNA–fluorogen complexes. In this study, we evaluated the feasibility of fluorescence correlation spectroscopy (FCS)- and fluorescence cross-correlation spectroscopy (FCCS)-based quantifications of FLAPs in a solution using Broccoli, a common FLAP, and its corresponding fluorogen, DFHBI-1T. We investigated the folding efficiency, photostability, and photophysical properties of the Broccoli–DFHBI-1T complex using their FCS/FCCS characteristics. With FCS, we observed that the fluorescence was affected by the affinity between Broccoli and DFHBI-1T and the folding (maturation) state of Broccoli RNA. Moreover, the FCCS measurement of ATTO647N-labeled Broccoli and its complex with DFHBI-1T revealed the proportion of the mature Broccoli–DFHBI-1T complex. The current FCS/FCCS-based study of Broccoli–DFHBI-1T provides a model for analyzing FLAPs and their fluorogen pairs at the single-molecule level

Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system.

Bioluminescent detection has become a powerful method that is used extensively in numerous areas in life science research. Given that fluorescence detection in plant cells is difficult owing to the autofluorescence of chlorophyll, the use of a luciferin-luciferase system should be effective in plant biology. However, the suitable optical window for a luminescence system in plants remains unexplored. In this study, we sought to determine the optical window and optimal luciferase reporter system for terrestrial plant analyses using Arabidopsis thaliana as a model organism. We compared six different luciferase systems and found the green enhanced Nano-lantern (GeNL)-furimazine combination to be the optimal luciferase reporter. Spectral measurements of GeNL-furimazine showed that its luminescence peak falls within the range of optical transparency for chlorophyll and, therefore, enables greater penetration through a layer of cultured A. thaliana cells. Moreover, A. thaliana plants expressing GeNL with furimazine emitted strong luminescence, which could be detected even with the naked eye. Thus, the GeNL-furimazine combination should facilitate biological analyses of genes and cellular functions in A. thaliana and all other terrestrial plants

Control of Adipogenic Differentiation in Mesenchymal Stem Cells via Endogenous Gene Activation Using CRISPR-Cas9

Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the <i>in vivo</i> differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue-specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system and achieved efficient induction of different types of adipocyte-like cells from MSCs. Interestingly, the MSCs converted via single-gene activation exhibited morphological and molecular properties of white adipocytes, while beige adipocyte-like cells were induced via multiplex gene activation of three specific transcription factors. These results reveal that the fate of MSCs can be effectively manipulated by direct activation of specific endogenous gene expression using a dCas9-based activator with reduced exogenous additives

Control of Adipogenic Differentiation in Mesenchymal Stem Cells via Endogenous Gene Activation Using CRISPR-Cas9

Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the <i>in vivo</i> differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue-specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system and achieved efficient induction of different types of adipocyte-like cells from MSCs. Interestingly, the MSCs converted via single-gene activation exhibited morphological and molecular properties of white adipocytes, while beige adipocyte-like cells were induced via multiplex gene activation of three specific transcription factors. These results reveal that the fate of MSCs can be effectively manipulated by direct activation of specific endogenous gene expression using a dCas9-based activator with reduced exogenous additives