Uptake Mechanism Of MNSC Derived Exosomes In The Neural Cells: Intracellular Communication Between MNSCs And The Neural Cells Via MNSC Derived Exosomes

Backgroound and Objectives:Retinitis pigmentosa(RP) is an inherited, degenerative eye disease that causes severe vision impairment due to the progressive degeneration of the rod photoreceptor cells in the retina. There is no cure for retinitis pigmentosa; however, the efficacy and safety of various prospective treatments are currently being evaluated. Transplantation of neural stem /precursor cells(NPCs)is one of the most promising methods. However, a comprehensive understanding of the mechanisms by which NPCs exert their therapeutic impact is lacking. It was first assumed that stem cells directly replace lost cells but it is now becoming clearer that they might be able to protect the nervous system through mechanisms other than cell replacement. Transplanted NPCs may also exert a ‘bystander’ neuroprotective effect and identified a series of molecules. Neural stem cell derived exosomes(EXOs) have been proposed to participate in modulation of the local microenvironment post transplantation of neural stem cell in neural degeneration diseases including retinitis pigmentosa. Extracellular Vesicles(EVs) are the extracellular membraneous nanovesicles, containing two major types of EVs, namely exosomes, and multi-Vesicles. They are key players in intercellular signaling. However,which kind of vesicles is involved in regulation of the host cells and is not fully understood yet. Although previous reports indicated that EVs could be mediated cell-to-cell interaction, how EVs interact with recipient cells remains unclear. In this study,we will focus on,1,Comparison of C17.2 neural stem cell derived Extracellular vesicles(EVs) and Exosomes were purified respectively by ultracentrifugation(EV) and Total Exosome Isolation kit(EXO);2,To study the intracellular communication between m NSCs and the neural cells via mNSC derived exosomes.Methods:1. C17.2 neural stem cell derived extracellular vesicles(EVs) and exosomes were purified respectively by ultracentrifugation(EV) and Total Exosome Isolation kit(EXO);2. Visualized under transmission electron microscopy(TEM) and Atomic Force Microscopy(AFM);3. The proteins of the two samples were displayed by SDS-PAGE electrophoresis, and specific marker proteins of exosomes were displayed by Western blot;4. Exosomes labeled by lipophilic dye;5. Exosomes incubated with 3T3-f GFP cells and neural cells including neuron, astrocyte, oligodendroglia to assay the uptake rate;6. To determine whether target cells internalize exosomes by endocytosis, different uptake pathway were selective blocked by various drugs.Results:1. C17.2 cells exhibited a well morphological structure did not appear obvious differentiation,indicated that EV derived from mNSCs.2. Bilayer liked membrane structure and the "cup" shape of the vesicles were often observed under EM, with about 300 nm diameters. The average diameter of EV was greater than the EXO.The rusult shows that besides exosomes the two samples contain other types of vesicles.3. According to the results of Atomic Force Microscopy, we observed the height of the sample more concentrated within 10-20 nm, lateral diameters range was about 300 nm. Both average height and average diameter of the EXO sample were bigger than the EV sample. The rusult shows that the two samples contain exosomes mainly4. SDS-PAGE results show that the two kinds of samples contain a variety of proteins. The protein amount is similar in two samples, but the different vesicle proportion is a bit different. This was further confirmed by specific exosomes marker probes. expression intensity were at similar level. Under the same sample content, HSP70 expression intensity was closed, but CD63, CD9 and CD81 expressed are found more abundant in EV sample.5. Exosomes were internalized respectively by 3T3-f GFP, primary glial cells, primary neuron and oligodendroglia through endocytosis,trapped in vesicles, and transported to perinuclear region, and the uptake exosomes were increased along times.6. The endocytosis not only can occur in the same species cells but also occur in different species cells.7. Clathrin-mediated endocytosis was the main pathway to uptake the exosome in 3 types of target cells.Conclusions:1. EV sample riched by ultracentrifugation is mainly containing exosomes, proved that the method is a simple, economic methods;2. 3T3-f GFP cells and 3 types of neural cells uptake exosomes through endocytosis with time relationship, different kinds of neural cells exist differences in number of internalization;3. 3T3-fGFP cells and 3 types of neural cells uptake exosomes through Clathrin-mediated endocytosis suggesting intracellular communication is possible between m NSCs and neural cells viap mNSC derived exosomes.