Advertisement

Journal of Molecular Medicine

, Volume 97, Issue 6, pp 829–844 | Cite as

ALIX increases protein content and protective function of iPSC-derived exosomes

  • Ruiting Sun
  • Yingying Liu
  • Meng Lu
  • Qianqian Ding
  • Pingping Wang
  • Heng Zhang
  • Xiaoyu Tian
  • Peng Lu
  • Dan Meng
  • Ning Sun
  • Meng XiangEmail author
  • Sifeng ChenEmail author
Original Article
  • 422 Downloads

Abstract

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.

Key messages

  • 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.

Keywords

Induced pluripotent stem cells Exosome Apoptosis-linked gene 2–interacting protein X Endothelial cells Endosomal proteomics 

Notes

Acknowledgments

We thank Malvern Instruments for technical support with the nano-particle tracking studies.

Author contributions

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.

Funding information

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.

Supplementary material

109_2019_1767_MOESM1_ESM.pdf (154 kb)
ESM 1 (PDF 154 kb)
109_2019_1767_MOESM2_ESM.pdf (38.8 mb)
ESM 2 (PDF 39702 kb)

References

  1. 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
  2. 2.
    Nakamori D, Takayama K, Nagamoto Y, Mitani S, Sakurai F, Tachibana M, Mizuguchi H (2016) Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPalpha, and PROX1 transduction. Biochem Biophys Res Commun 469(3):424–429CrossRefGoogle Scholar
  3. 3.
    Umeda K, Shiraki N, Kume S (2016) Hepatic differentiation from human Ips cells using M15 cells. Methods Mol Biol 1357:375–381CrossRefGoogle Scholar
  4. 4.
    Ebben JD, Zorniak M, Clark PA, Kuo JS (2011) Introduction to induced pluripotent stem cells: advancing the potential for personalized medicine. World Neurosurg 76(3–4):270–275CrossRefGoogle Scholar
  5. 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. 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. 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
  8. 8.
    Choi DS, Kim DK, Kim YK, Gho YS (2015) Proteomics of extracellular vesicles: exosomes and ectosomes. Mass Spectrom Rev 34(4):474–490CrossRefGoogle Scholar
  9. 9.
    Thery C (2011) Exosomes: secreted vesicles and intercellular communications. F1000 Biol Rep 3:15.  https://doi.org/10.3410/B3-15 CrossRefGoogle Scholar
  10. 10.
    Tao SC, Yuan T, Zhang YL, Yin WJ, Guo SC, Zhang CQ (2017) Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model. Theranostics 7(1):180–195CrossRefGoogle Scholar
  11. 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
  12. 12.
    Ong SG, Lee WH, Huang M, Dey D, Kodo K, Sanchez-Freire V, Gold JD, Wu JC (2014) Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer. Circulation 130(11 Suppl 1):S60–S69CrossRefGoogle Scholar
  13. 13.
    Vandergriff A, Huang K, Shen D, Hu S, Hensley MT, Caranasos TG, Qian L, Cheng K (2018) Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide. Theranostics 8(7):1869–1878CrossRefGoogle Scholar
  14. 14.
    Tickner JA, Urquhart AJ, Stephenson SA, Richard DJ, O'Byrne KJ (2014) Functions and therapeutic roles of exosomes in cancer. Front Oncol 4:127.  https://doi.org/10.3389/fonc.2014.00127 CrossRefGoogle Scholar
  15. 15.
    Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, Wieland F, Schwille P, Brugger B, Simons M (2008) Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319(5867):1244–1247CrossRefGoogle Scholar
  16. 16.
    Henne WM, Buchkovich NJ, Emr SD (2011) The ESCRT pathway. Dev Cell 21(1):77–91CrossRefGoogle Scholar
  17. 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
  18. 18.
    Dowlatshahi DP, Sandrin V, Vivona S, Shaler TA, Kaiser SE, Melandri F, Sundquist WI, Kopito RR (2012) ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus budding. Dev Cell 23(6):1247–1254CrossRefGoogle Scholar
  19. 19.
    Matsuo H, Chevallier J, Mayran N, Le Blanc I, Ferguson C, Faure J, Blanc NS, Matile S, Dubochet J, Sadoul R, Parton RG, Vilbois F, Gruenberg J (2004) Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 303(5657):531–534CrossRefGoogle Scholar
  20. 20.
    Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E, Zimmermann P, David G (2012) Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 14(7):677–685CrossRefGoogle Scholar
  21. 21.
    Hemming FJ, Fraboulet S, Blot B, Sadoul R (2004) Early increase of apoptosis-linked gene-2 interacting protein X in areas of kainate-induced neurodegeneration. Neuroscience 123(4):887–895CrossRefGoogle Scholar
  22. 22.
    Chatellard-Causse C, Blot B, Cristina N, Torch S, Missotten M, Sadoul R (2002) Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J Biol Chem 277(32):29108–29115CrossRefGoogle Scholar
  23. 23.
    Sun N, Panetta NJ, Gupta DM, Wilson KD, Lee A, Jia F, Hu S, Cherry AM, Robbins RC, Longaker MT, Wu JC (2009) Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci U S A 106(37):15720–15725CrossRefGoogle Scholar
  24. 24.
    Xiang M, Lu M, Quan J, Xu M, Meng D, Cui A, Li N, Liu Y, Lu P, Kang X, Wang X, Sun N, Zhao M, Liang Q, Le L, Wang X, Zhang J, Chen S (2019) Direct in vivo application of induced pluripotent stem cells is feasible and can be safe. Theranostics 9(1):290–310CrossRefGoogle Scholar
  25. 25.
    Cui A, Xiang M, Xu M, Lu P, Wang S, Zou Y, Qiao K, Jin C, Li Y, Lu M, Chen AF, Chen S (2018) VCAM-1-mediated neutrophil infiltration exacerbates ambient fine particle-induced lung injury. Toxicol Lett 302:60–74CrossRefGoogle Scholar
  26. 26.
    Jiang L, Yin M, Wei X, Liu J, Wang X, Niu C, Kang X, Xu J, Zhou Z, Sun S, Wang X, Zheng X, Duan S, Yao K, Qian R, Sun N, Chen A, Wang R, Zhang J, Chen S, Meng D (2015) Bach1 represses Wnt/beta-catenin signaling and angiogenesis. Circ Res 117(4):364–375CrossRefGoogle Scholar
  27. 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
  28. 28.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308CrossRefGoogle Scholar
  29. 29.
    Wang J, Yan S, Zhang W, Zhang H, Dai J (2015) Integrated proteomic and miRNA transcriptional analysis reveals the hepatotoxicity mechanism of PFNA exposure in mice. J Proteome Res 14(1):330–341CrossRefGoogle Scholar
  30. 30.
    Falguières T, Luyet PP, Bissig C, Scott CC, Velluz MC, Gruenberg J (2008) In vitro budding of intralumenal vesicles into late endosomes is regulated by Alix and Tsg101. Mol Biol Cell 19(11):4942–4955CrossRefGoogle Scholar
  31. 31.
    Boucrot E, Ferreira AP, Almeida-Souza L, Debard S, Vallis Y, Howard G, Bertot L, Sauvonnet N, McMahon HT (2015) Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517(7535):460–465CrossRefGoogle Scholar
  32. 32.
    Rahajeng J, Giridharan SS, Naslavsky N, Caplan S (2010) Collapsin response mediator protein-2 (Crmp2) regulates trafficking by linking endocytic regulatory proteins to dynein motors. J Biol Chem 285(42):31918–31922CrossRefGoogle Scholar
  33. 33.
    Martins AS, Ordonez JL, Amaral AT, Prins F, Floris G, Debiec-Rychter M, Hogendoorn PC, de Alava E (2011) IGF1R signaling in Ewing sarcoma is shaped by clathrin−/caveolin-dependent endocytosis. PLoS One 6(5):e19846.  https://doi.org/10.1371/journal.pone.0019846 CrossRefGoogle Scholar
  34. 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
  35. 35.
    Keerthikumar S, Chisanga D, Ariyaratne D, Al Saffar H, Anand S, Zhao K, Samuel M, Pathan M, Jois M, Chilamkurti N, Gangoda L, Mathivanan S (2016) ExoCarta: a web-based compendium of exosomal cargo. J Mol Biol 428(4):688–692CrossRefGoogle Scholar
  36. 36.
    Kapustin AN, Chatrou MLL, Drozdov I, Zheng Y, Davidson SM, Soong D, Furmanik M, Sanchis P, Rosales RTMD, Alvarezhernandez D (2015) Vascular smooth muscle cell calcification is mediated by regulated exosome secretion. Circ Res 116(8):1312–1323CrossRefGoogle Scholar
  37. 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
  38. 38.
    Camussi G, Deregibus MC, Cantaluppi V (2013) Role of stem-cell-derived microvesicles in the paracrine action of stem cells. Biochem Soc Trans 41(1):283–287CrossRefGoogle Scholar
  39. 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
  40. 40.
    Greening DW, Gopal SK, Xu R, Simpson RJ, Chen W (2015) Exosomes and their roles in immune regulation and cancer. Semin Cell Dev Biol 40:72–81CrossRefGoogle Scholar
  41. 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
  42. 42.
    Hu GW, Li Q, Niu X, Hu B, Liu J, Zhou SM, Guo SC, Lang HL, Zhang CQ, Wang Y, Deng ZF (2015) Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. Stem Cell Res Ther 6:10CrossRefGoogle Scholar
  43. 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
  44. 44.
    Barile L, Milano G, Vassalli G (2017) Beneficial effects of exosomes secreted by cardiac-derived progenitor cells and other cell types in myocardial ischemia. Stem Cell Investig 4:93CrossRefGoogle Scholar
  45. 45.
    van Niel G, Charrin S, Simoes S, Romao M, Rochin L, Saftig P, Marks MS, Rubinstein E, Raposo G (2011) The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis. Dev Cell 21(4):708–721CrossRefGoogle Scholar
  46. 46.
    Haucke V (2015) Cell biology: on the endocytosis rollercoaster. Nature 517(7535):446–447CrossRefGoogle Scholar
  47. 47.
    Prosser DC, Tran D, Schooley A, Wendland B, Ngsee JK (2010) A novel, retromer-independent role for sorting nexins 1 and 2 in RhoG-dependent membrane remodeling. Traffic 11(10):1347–1362CrossRefGoogle Scholar
  48. 48.
    Caponnetto F, Manini I, Skrap M, Palmai-Pallag T, Di Loreto C, Beltrami AP, Cesselli D, Ferrari E (2017) Size-dependent cellular uptake of exosomes. Nanomedicine 13(3):1011–1020CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Physiology and Pathophysiology, School of Basic Medical SciencesFudan UniversityShanghaiChina

Personalised recommendations