Primed mesenchymal stem cell-derived exosomes as a potential therapeutic strategy for inflammatory arthritis
Primed mesenchymal stem cell-derived exosomes as a potential therapeutic strategy for inflammatory arthritis
- 주제(키워드) Mesenchymal stem cells (MSCs) , Exosomes , Priming , Inflammatory arthritis , Three-dimensional (3D)
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028019
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Mesenchymal stem cells (MSCs) have emerged as a good source of cells for therapeutic applications. The therapeutic efficacy of MSCs has been demonstrated for a variety of target diseases, and MSCs have already obtained market approval in certain countries. The therapeutic efficacy of MSCs is attributed mainly to their paracrine secretion of cytokines and growth factors, which exert diverse cellular activities, such as immune modulation, anti-inflammatory effects, angiogenic effects, and neurogenesis. However, studies have shown that MSCs exert their paracrine effects by secreting not only trophic factors, but also exosomes. Exosomes are secreted vesicles of endocytic origin that are 55–100 nm in size and secreted by a variety of cells types, including MSCs. Exosomes mediate local or systemic cell-to-cell communication and alter cellular metabolism through the transfer of various proteins, mRNAs, and microRNAs (miRNAs). Recent studies have shown that MSC-derived exosomes mediate the therapeutic effects of MSCs, and suggest their potential as a cell-free therapy because of their lower immunogenicity, and ease of manipulation and storage, as compared to cell-based products. For example, MSC-derived exosomes have been reported to be therapeutically active for the treatment of osteoarthritis (OA); however, their ability to inhibit inflammation in OA has not yet been reported. Therefore, in this study we aimed develop exosomes as a therapeutic agent for inflammatory arthritis by improving their anti-inflammatory properties and increasing their production by priming MSCs. Chapter 1 describes our investigation of the anti-inflammatory effect and mechanism of exosomes, that were derived from MSCs primed with Interleukin-1 beta (IL-1β) (MSCs-IL-Exo), in SW982 human synovial cells treated with IL-1β and tumor necrosis factor alpha (TNF-α). We demonstrated that exosomes isolated from both unprimed and IL-1β-primed MSCs were round in shape and approximately 30-300 nm in size. Exosomes from both IL-1β-primed and unprimed MSCs also expressed CD9, CD63, and CD81 at similar levels, as shown by Western blot analysis. In the inflammatory model of SW982 cells induced by IL-1β and TNF-α, MSCs-IL-Exo showed higher inhibitory activity against IL-1β, IL-6, and MCP-1 than MSCs from unprimed MSCs (MSCs-Exo). Profiling of small RNAs demonstrated that the priming of IL-1β altered small RNA levels in exosomes secreted from MSCs. Moreover, among the anti-inflammatory miRNAs we examined, miRNA 147b (miR-147b) was present at higher levels in MSCs-IL-Exo than in MSC-Exo. Moreover, we found that transfecting inflammatory SW982 cells with miR-147b led to inhibition of IκBα degradation. These results suggest that MSCs-IL-Exo are more efficacious in inhibiting NFκB activation than MSCs-Exo, and that miR-147b shuttled by MSCs-IL-Exo may be involved. Chapter II describes our investigation of whether three-dimensional (3D) spheroid culture of MSCs improves the efficiency of exosome production and if so, the underlying mechanism. We adopted two models of 3D-spheroid culture using the hanging-drop (3D-HD) and poly 2 hydroxyethyl methacrylate (poly-HEMA) coating methods (3D-PH). The 3D-HD method formed a single large spheroid, while the 3D-PH method formed multiple smaller ones. MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional two-dimensional (2D) monolayer cultures. We then investigated the effects of hypoxia, high cell density, and non-adherent cell morphology on exosome production. We observed that increased spheroid size was associated with greater exosome production, while the 3D-HD and 3D-PH methods produced the fewest cells. Increasing the cell density in 2D culture (2D-H) led to less efficient exosome production than conventional, lower cell density 2D culture. Finally, plating cells at a normal density on poly-HEMA coated spheroids (termed 3D-N-PH), led to the formation of small aggregates consisting of less than ten cells that produced more exosomes than 2D-cultured cells plated at the same density. Moreover, F-actin expression was markedly reduced in the 3D-N-PH culture. Together, these results suggest that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent rounded cell morphology itself might be a causative factor. These results could provide useful information for developing an optimal process for the mass production of exosomes.
more목차
ACKNOWLEDGEMENTS i
ABSTRACT iii
LIST OF FIGURES vii
LIST OF TABLES viii
LIST OF ABBREVIATIONS ix
BACKGROUND 1
1.1. Mesenchymal stem/stromal cell (MSC): Relevance in regenerative medicine 2
1.2. Exosome: Small vesicles participating in the cell-to-cell communication 4
1.3. Exosome derived from MSCs as a novel approach to cell-free therapy 5
1.4. Priming factors of Exosome 6
1.5. Aim of the study 8
CHAPTER I: Exosomes derived from IL-1β -primed MSCs inhibit IL-1β and TNF-α-mediated inflammatory response in SW982 cells 9
2.1.Introduction 10
2.2.Materials and Methods 12
2.2.1.Cell culture 12
2.2.2.Conditioned media (CM) preparation 12
2.2.3.Exosome isolation 12
2.2.4.Exosome characterization and internalization analysis 12
2.2.5.RNase-digested exosomes preparation 13
2.2.6.Anti-inflammation assay 13
2.2.7.RNA analysis 13
2.2.8.Transfection of miRNA mimic and inhibitor 13
2.2.9.RNA analysis 14
2.2.10.Western blot analysiss 14
2.2.11.Statistical analysis 14
2.3.Results 16
2.3.1.Exosomes derived from MSCs (MSCs-Exo) and IL-1β-primed MSCs (MSCs-IL-Exo) have similar characteristics 16
2.3.2.MSCs-IL-Exo showed higher ability to inhibit inflammatory events in inflammatory arthritis like SW982 18
2.3.3.Anti-inflammatory effect of MSCs-IL-Exo is probably mediated via exosome-shuttled RNA 20
2.3.4.MicroRNA 147b (miR-147b) contributed to the anti-inflammatory effect of MSCs-IL-Exo on inflammatory arthritis like SW982 23
2.3.5.Anti-inflammatory effect of MSCs-IL-Exo involved NFκB activation 25
2.4.Discussion 28
CHAPTER II: Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells 31
3.1.Introduction 32
3.2.Materials and Methods 32
3.2.1.Cell culture 34
3.2.2.Preparation of experimental groups 34
3.2.3.Characterization of cell morphology and spheroids size using Image J 34
3.2.4.Isolation of exosomes 35
3.2.5.Western blot analysis 35
3.2.6.Statistical analysis 35
3.3.Results 36
3.3.1.hBM-MSCs in 3D spheroid culture secreted more exosomes than those in 2D culture 36
3.3.2.The production efficiency of exosomes from hBM-MSCs decreased with increasing 3D spheroid size 38
3.3.3.The production efficiency of exosomes decreased with increasing cell density in 2D culture of hBM-MSCs 40
3.3.4.The non-adherent state of cell morphology might be important in the enhanced production efficiency of exosomes from hBM-MSCs 42
3.4.Discussion 46
CONCLUSIONS 48
REFERENCES 50
