Abstract
Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mendicino M, Bailey AM, Wonnacott K et al (2014) MSC-based product characterization for clinical trials: an FDA perspective. Cell Stem Cell 14:141–145
Lai RC, Chen TS, Lim SK (2011) Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease. Regen Med 6:481–492
Gnecchi M, He H, Noiseux N et al (2006) Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 20:661–669
Gnecchi M, He H, Noiseux N et al (2005) Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med 11:367–368
Mirotsou M, Zhang Z, Deb A et al (2007) Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. Proc Natl Acad Sci U S A 104:1643–1648
Timmers L, Lim SK, Arslan F et al (2007) Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. Stem Cell Res 1:129–137
Lai RC, Arslan F, Lee MM et al (2010) Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 4:214–222
Lai RC, Arslan F, Tan SS et al (2010) Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles. J Mol Cell Cardiol 48:1215–1224
Bi B, Schmitt R, Israilova M et al (2007) Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol 18:2486–2496
Parekkadan B, van Poll D, Suganuma K et al (2007) Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS One 2, e941
Timmers L, Lim SK, Hoefer IE et al (2011) Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction. Stem Cell Res 6:206–214
Schafer R, Northoff H (2008) Cardioprotection and cardiac regeneration by mesenchymal stem cells. Panminerva Med 50:31–39
Haynesworth SE, Baber MA, Caplan AI (1996) Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol 166:585–592
Kinnaird T, Stabile E, Burnett MS et al (2004) Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences. Circ Res 95:354–363
Patschan D, Plotkin M, Goligorsky MS (2006) Therapeutic use of stem and endothelial progenitor cells in acute renal injury: ca ira. Curr Opin Pharmacol 6:176–183
Miyahara Y, Nagaya N, Kataoka M et al (2006) Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 12:459–465
Min JY, Sullivan MF, Yang Y et al (2002) Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs. Ann Thorac Surg 74:1568–1575
Kinnaird T, Stabile E, Burnett MS et al (2004) Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94:678–685
Sze SK, de Kleijn DP, Lai RC et al (2007) Elucidating the secretion proteome of human embryonic stem cell-derived mesenchymal stem cells. Mol Cell Proteomics 6:1680–1689
Kordelas L, Rebmann V, Ludwig AK et al (2014) MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 28:970–973
Ludwig AK, Kordelas L, Rebmann V et al (2012) Exosomes – from bench to bedside. Klin Padiatr 224:A6
Xin H, Li Y, Buller B et al (2012) Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem Cells 30:1556–1564
Li T, Yan Y, Wang B et al (2013) Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev 22:845–854
Lee C, Mitsialis SA, Aslam M et al (2012) Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension. Circulation 126:2601–2611
Reis LA, Borges FT, Simões MJ et al (2012) Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats. PLoS One 7:e44092
Tan CY, Lai RC, Wong W et al (2014) Mesenchymal stem cell-derived exosomes promote hepatic regeneration in drug-induced liver injury models. Stem Cell Res Ther 5:76, Epub ahead of print
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33:967–978
Chen TS, Arslan F, Yin Y et al (2011) Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J Transl Med 9:47
Acknowledgments
This work was supported by funding from A*STAR. We thank members of our laboratories for their contribution to this work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Appendix: Purification Method
Appendix: Purification Method
Main method:
¤ (Main)
0.00 Base CV (26.5)#Column_volume [31] Any
0.00 Flow 1 {mL/min}
0.00 Watch_Pressure Greater_Than 2.9 [32] PAUSE
0.00 BufferValveA1 A12
0.00 ColumnPosition Position3
0.00 Alarm_Pressure Enabled 2.9 [32] 0.00 [32]
0.00 Wavelength 220 {nm} 280 {nm} 260 {nm}
¤ 0.00 Block Equilibration
(Equilibration)
0.00 Base SameAsMain
0.5 AutoZeroUV
0.50 End_Block
¤ 0.00 Block Sample_injection
(Sample_injection)
0.00 Base Volume
0.00 InjectionValve Inject
1.5 InjectionValve (Load)#Load_volume
1.50 End_Block
¤ 0.00 Block Fractionation
(Fractionation)
0.00 Base SameAsMain
0.25 OutletValve F2
0.25 Fractionation 12 mm 0.5 [31] FirstTube Volume
0.55 FractionationStop
0.55 OutletValve WasteF1
1.10 End_Block
¤ 0.00 Block Reequilibration
(Reequilibration)
0.00 Base SameAsMain
0.5 End_Block
0.00 End_method
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lai, R.C., Yeo, R.W.Y., Padmanabhan, J., Choo, A., de Kleijn, D.P.V., Lim, S.K. (2016). Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells. In: Gnecchi, M. (eds) Mesenchymal Stem Cells. Methods in Molecular Biology, vol 1416. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3584-0_29
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3584-0_29
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3582-6
Online ISBN: 978-1-4939-3584-0
eBook Packages: Springer Protocols