ALIX increases protein content and protective function of iPSC-derived exosomes
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Nature of exosome-secreting cells determines exosome content and function. ALIX, involved in exosome biogenesis, promotes cell degeneration. Here, ALIX was knocked out (iPSC-ALIX−/−) and overexpressed (iPSC-ALIX3+) in induced pluripotent stem cells (iPSCs) using CRISPR-Cas9 and lentiviral transduction, respectively, and the secreted exosomes were analyzed. Exosomes from iPSC-ALIX−/− (exosome-KO), iPSC-ALIX3+ (exosome-over), and their corresponding controls contained 176, 529, 431, and 351 proteins, respectively. Exosome-over showed increased protein levels, while exosome-KO contained fewer protein types without differing in total protein content. ALIX knockout did not affect exosome uptake by endothelial cells. Exosome-over more effectively promoted cell viability than exosome-GFP, in a dose-dependent manner. All exosomes were protective for endothelial cells injured by hydrogen peroxide or cisplatin, as demonstrated by promotion of cell viability, horizontal migration, angiogenic sprouting from aortic rings, and formation of capillary-like structures, inhibition of apoptosis, and maintenance of permeability of endothelial monolayer, although exosome-over and exosome-KO had stronger and weaker effects, respectively. SNX2 was important for ALIX-mediated exosomal function. Beneficial functions of the exosomes were independent of experimental models, targeted cell types, causes of injury, exosome-producing iPSC passages, clones of ALIX knockout, and transfection batches of ALIX overexpression. Thus, we present a novel strategy to manipulate iPSCs for production of exosomes with beneficial ALIX-regulated protein composition for varied exosome functions.
ALIX knockout and overexpression regulate protein profile in iPSC-derived exosome.
ALIX knockout decreases therapeutic function of iPSC-derived exosomes.
ALIX overexpression increases therapeutic function of iPSC-derived exosomes.
Manipulating iPSCs can produce exosomes with more beneficial protein content.
KeywordsInduced pluripotent stem cells Exosome Apoptosis-linked gene 2–interacting protein X Endothelial cells Endosomal proteomics
We thank Malvern Instruments for technical support with the nano-particle tracking studies.
R.S., Y.L., M. L, P.L., M.X., X.T., and Q.D.: data collection. P.W., H.Z., D.M., and N.S.: data analysis. S.C.: conception and design, data analysis and interpretation, financial support, administrative support, manuscript writing, final approval of manuscript.
This study was supported by Great Research Plan Program (91539120 to S. Chen), International Cooperation and Exchanges (81220108002 to S. Chen), and General Program (81470260 to M. Xiang) of the National Natural Science Foundation of China, and the National Key R&D Program of China (2016YFC1305101 to S. Chen).
Compliance with ethical standards
The animal protocol for mouse aorta collection was approved by the Animal Care Committee of the Fudan University Shanghai Medical College in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council of USA). Three fresh umbilical cord veins were obtained from women with normal pregnancies and delivery after informed consent was obtained to isolate primary HUVECs with the approval of the Ethics Board of Fudan University Shanghai College of Medicine in accordance with the Helsinki Declaration of 1975.
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.Ye L, Chang YH, Xiong Q, Zhang P, Zhang L, Somasundaram P, Lepley M, Swingen C, Su L, Wendel JS, Guo J, Jang A, Rosenbush D, Greder L, Dutton JR, Zhang J, Kamp TJ, Kaufman DS, Ge Y, Zhang J (2014) Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell 15(6):750–761CrossRefGoogle Scholar
- 5.Kikuchi T, Morizane A, Doi D, Magotani H, Onoe H, Hayashi T, Mizuma H, Takara S, Takahashi R, Inoue H, Morita S, Yamamoto M, Okita K, Nakagawa M, Parmar M, Takahashi J (2017) Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model. Nature 548(7669):592–596CrossRefGoogle Scholar
- 6.Ding QQ, Sun RT, Wang PP, Zhang H, Xiang M, Meng D, Sun N, Chen FY, Chen SF (2016) Protective effects of human induced pluripotent stem cell-derived exosomes on high glucose-induced injury in human endothelial cells. Exp Ther Med 32(8)Google Scholar
- 7.Zhang J, Guan J, Niu X, Hu G, Guo S, Li Q, Xie Z, Zhang C, Wang Y (2015) Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med 13:49. https://doi.org/10.1186/s12967-015-0417-0 CrossRefGoogle Scholar
- 11.Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, Mackie AR, Vaughan E, Garikipati VN, Benedict C, Ramirez V, Lambers E, Ito A, Gao E, Misener S, Luongo T, Elrod J, Qin G, Houser SR, Koch WJ, Kishore R (2015) Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circ Res 117(1):52–64CrossRefGoogle Scholar
- 17.Keren-Kaplan T, Attali I, Estrin M, Kuo LS, Farkash E, Jerabek-Willemsen M, Blutraich N, Artzi S, Peri A, Freed EO, Wolfson HJ, Prag G (2013) Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding. EMBO J 32(4):538–551CrossRefGoogle Scholar
- 27.Deshane J, Chen S, Caballero S, Grochot-Przeczek A, Was H, Li Calzi S, Lach R, Hock TD, Chen B, Hill-Kapturczak N, Siegal GP, Dulak J, Jozkowicz A, Grant MB, Agarwal A (2007) Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism. J Exp Med 204(3):605–618CrossRefGoogle Scholar
- 34.Perin EC, Sanz-Ruiz R, Sanchez PL, Lasso J, Perez-Cano R, Alonso-Farto JC, Perez-David E, Fernandez-Santos ME, Serruys PW, Duckers HJ, Kastrup J, Chamuleau S, Zheng Y, Silva GV, Willerson JT, Fernandez-Aviles F (2014) Adipose-derived regenerative cells in patients with ischemic cardiomyopathy: the PRECISE trial. Am Heart J 168(1):88–95 e82CrossRefGoogle Scholar
- 37.Hong KU, Guo Y, Li QH, Cao P, Al-Maqtari T, Vajravelu BN, Du J, Book MJ, Zhu X, Nong Y, Bhatnagar A, Bolli R (2014) C-kit+ cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart. PLoS One 9(5):e96725. https://doi.org/10.1371/journal.pone.0096725 CrossRefGoogle Scholar
- 39.Otsuru S, Gordon PL, Shimono K, Jethva R, Marino R, Phillips CL, Hofmann TJ, Veronesi E, Dominici M, Iwamoto M (2012) Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms. Blood 120(9):1933–1941CrossRefGoogle Scholar
- 41.da Silva Lara L, Andrade-Lima L, Calvet CM, Borsoi J, Alberto Duque TL, Henriques-Pons A, Souza Pereira MC, Pereira LV (2018; [Epub ahead of print]) Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease. Microbes Infect doi:, 20, 312, 316Google Scholar
- 43.Wang Y, Zhang L, Li Y, Chen L, Wang X, Guo W, Zhang X, Qin G, He SH, Zimmerman A, Liu Y, Kim IM, Weintraub NL, Tang Y (2015) Exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium. Int J Cardiol 192:61–69CrossRefGoogle Scholar