학위논문 (박사)-- 서울대학교 융합과학기술대학원 : 융합과학기술대학원 분자의학및바이오제약학과, 2016. 2. 이동수.Neuronal cells release small vesicles known as secretory exosomes containing mRNAs, miRNAs and proteins to exchange signals as a form of intercommunication between cells. MicroRNAs (miRNAs) such as miR-124 or miR-9 play an important role in regulation of neuronal differentiation. Intercellular transfer of neurogenic microRNA (miRNA) induces neurogenesis and exosomes can mediate miRNA delivery from the leading differentiated cells to neighboring undifferentiated cells. The aim of this study is to confirm cell-non-autonomous miRNA/exosome-mediated differentiation of neural progenitor cells and to visualize exosomes carrying this neurogenic miRNA from leading to neighboring cells. F11 cells, neural progenitor cells, were stably transfected with reporter vector of pRV-effLuc/3xPT_miR-193a which luciferase signal could be turned off by binding of the identified miRNA to the triplicates of miRNA binding site in the 3' UTR of effLuc. Exosomes were isolated from the conditioned media and characterized by western blot. Transwell chambers system and microfluidic device were used to examine exosome-mediated miRNA transfer. The CMV-driven GFP-tagged CD63 vector was used to visualize endogenous exosomes. Target genes of MiR-193a considered as neurogenic miRNAs were related to cell proliferation, differentiation and axon guidance. Neurite outgrowth and neuronal marker expression such as βIII-tubulin, NeuroD and MAP2 were observed 3 days after identified MiR-193a treatment. Isolated exosomes were characterized for protein markers such as CD63, TSG101. qRT-PCR results showed that exosomes isolated from conditioned media in differentiated F11 cells highly increased miR-193a level. In addition to 2D co-culture and transwell culture setting, fluorescence signals of incorporated GFP-exosomes were detected within 2 days after co-culture with GFP-exosomes producing cells. Time-lapse live-cell confocal imaging using microfluidic device visualized the transport of single exosomes from differentiated to undifferentiated F11 cells. MiR-193a within the exosomes from differentiated donor F11 cells reached the recipient cells and was taken up to lead them to neuronal differentiation, showing increased neuronal marker expression. And these phenomenon were also reproduced in NE-4C, neural stem cells. Inhibition of the exosomal production by manumycin-A and treatment of anti-miR-193a in the differentiated donor cells failed to induce neurogenesis in undifferentiated recipient cells. These findings indicate that exosomes of neural progenitors and neurogenic miRNA within these exosomes propagate cell-non-autonomous differentiation to neighboring progenitors, which was captured visually on microfluidic device to delineate the roles of extracellular vesicles mediating neurogenesis of population of homologous progenitor cells.INTRODUCTION 12
1) Extracellular vesicles (EVs) 13
2) MicroRNA (miRNA) 15
3) Neurogenesis 18
4) MiRNA microarray 19
5) Co-culture system 19
6) MiRNA optical reporter gene 21
MATERIALS AND METHODS 23
Microarray Analysis 23
Gene Annotation Analysis 24
Cell culture and transfection 24
Quantitative reverse transcription-PCR 26
Immunofluorescence staining 26
In vitro luciferase assay 28
Preparation and characterization of exosomes 28
Fluorescent labeling of exosomes and cells 28
Transwell assay 29
Western blotting 30
Microfluidic cell culture device 31
Time-lapse imaging of exosomes 32
Bioluminescence imaging 33
Statistical analysis 33
Results 34
Identification of miR-193a newly associated with neurogenesis in Ngn1-overexpressing F11 cells 34
Enhanced neuronal differentiation in neural progenitor cells by overproduction of miR-193a 35
Elevated levels of miR-193a in differentiated F11 cell-derived exosomes 37
Exosomes-mediated transfer of miR-193a from differentiated cells to undifferentiated cells 38
Visualization of transport of individual exosomes using a microfluidic device 41
Reporting the action of exosomally delivered miR-193a in recipient F11-UD cells on microfluidic chamber 42
Discussion 84
Reference 92
Abstract in Korean 104Docto
