Optimization of Protein Quantification in Wharton Jelly-Derived Mesenchymal Stem Cell Exosomes

Yıl 2023, Cilt: 6 Sayı: 3, 452 - 458, 21.10.2023

Zehra Seda Ünal Halbutoğulları , Zeynep Ece Utkan Korun , Cansu Subaşı Demir , Kamil Can Kılıç , Büşra Alper Yusufhan Yazır

Öz

Aim: Exosomes are small intracellular membrane-based vesicles with different compositions that are involved in various biological and pathological processes. They are secreted by all cell types and can be found in most body fluids, including the blood, saliva, and urine. Exosomes are nanometer-sized microvesicles, approximately 30-200 nm in diameter, that contain DNA, mRNAs, non-coding RNAs, membrane proteins, and cytosolic proteins. Mesenchymal stem cells exert paracrine effects through exosomes. Recent studies have shown that exosomes have important potential as a new alternative to cellular therapies. In this study, we aimed to obtain exosomes from Wharton-Jelly derived mesenchymal stem cells (WJ-MSCs), characterize methods, quantify protein from these exosomes, and optimize this by comparing it with the amount of nanoparticles and determining the most effective lysis solution using different lysis chemicals.
Methods: For this purpose, first, WJ-MSCs' exosomes were isolated and characterized, and changes in protein levels were determined after treatment with 1 billion/ml particle exosomes with commonly used chemicals such as RIPA buffer, Mammalian Protein Extraction Reagent (M-PER), Tris-TritonX, and Tris-SDS.
Results: As a result, approximately 2.5 µg/ml of 1 billion/ml particulate exosomes were detected with the BCA kit. Concentration of protein in these exosomes, and the protein concentration increased 3-4 times as a result of disintegration of the bilayer membranes in the cell membrane structure of these exosomes with RIPA buffer.
Conclusion: It is predicted that these data can be used comparatively in future studies, especially for the quantification of mesenchymal stem cell exosomes.

Anahtar Kelimeler

Wharton Jelly, Exosome, Mesenchymal Stem Cell, Protein Quantification, Lysis Buffer

Destekleyen Kurum

Kocaeli Üniversitesi-BAP

Proje Numarası

TMP-2022-2352

Kaynakça

• Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 2007;25(6):1384-1392. doi:10.1634/stemcells.2006-0709
• Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal. 2011;9:1-14. doi:10.1186/1478-811X-9-12
• Mennan C, Wright K, Bhattacharjee A, Balain B, Richardson J, Roberts S. Isolation and characterisation of mesenchymal stem cells from different regions of the human umbilical cord. Biomed Res Int. 2013;2013:916136. doi:10.1155/2013/916136
• Semenova E, Grudniak MP, Machaj EK, et al. Mesenchymal stromal cells from different parts of umbilical cord: approach to comparison & characteristics. Stem Cell Rev Reports. 2021;17(5):1780-1795. doi:10.1007/s12015-021-10157-3
• Weiss ML, Troyer DL. Stem cells in the umbilical cord. Stem Cell Rev. 2006;2(2):155-162. doi:10.1007/s12015-006-0022-y
• Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317. doi:10.1080/14653240600855905
• Krampera M, Galipeau J, Shi Y, Tarte K, Sensebe L. Immunological characterization of multipotent mesenchymal stromal cells-The international society for cellular therapy (ISCT) working proposal. Cytotherapy. 2013;15(9):1054-1061. doi:10.1016/j.jcyt.2013.02.010
• Batsali AK, Kastrinaki M, Papadaki HA, Pontikoglou C. Mesenchymal stem cells derived from Wharton’s Jelly of the umbilical cord: biological properties and emerging clinical applications. Curr Stem Cell Res Ther. 2013;8(2):144-55. doi:10.2174/1574888x11308020005
• Kim DW, Staples M, Shinozuka K, Pantcheva P, Kang SD, Borlongan C V. Wharton’s jelly-derived mesenchymal stem cells: Phenotypic characterization and optimizing their therapeutic potential for clinical applications. Int J Mol Sci. 2013;14(6):11692-11712. doi:10.3390/ijms140611692
• Lai RC, Yeo RWY, Tan KH, Lim SK. Exosomes for drug delivery - A novel application for the mesenchymal stem cell. Biotechnol Adv. 2013;31(5):543-551. doi:10.1016/j.biotechadv.2012.08.008
• Kooijmans SAA, Vader P, van Dommelen SM, van Solinge WW, Schiffelers RM. Exosome mimetics: A novel class of drug delivery systems. Int J Nanomedicine. 2012;7:1525-1541. doi:10.2147/IJN.S29661
• Schiller M, Bekeredjian-Ding I, Heyder P, Blank N, Ho AD, Lorenz HM. Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis. Cell Death Differ. 2008;15(1):183-191. doi:10.1038/sj.cdd.4402239
• Ha D, Yang N, Nadithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm Sin B. 2016;6(4):287-296. doi:10.1016/j.apsb.2016.02.001
• Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem. 2019;88:487-514. doi:10.1146/annurev-biochem-013118-111902
• Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1). doi:10.1080/20013078.2018.1535750
• Bian D, Wu Y, Song G, Azizi R, Zamani A. The application of mesenchymal stromal cells (MSCs) and their derivative exosome in skin wound healing: a comprehensive review. Stem Cell Res Ther. 2022;13(1):24. doi:10.1186/s13287-021-02697-9
• Zhang Y, Bi J, Huang J, Tang Y, Du S, Li P. Exosome: A review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications. Int J Nanomedicine. 2020;15:6917-6934. doi:10.2147/IJN.S264498
• Zhou W, Silva M, Feng C, et al. Exosomes derived from human placental mesenchymal stem cells enhanced the recovery of spinal cord injury by activating endogenous neurogenesis. Stem Cell Res Ther. 2021;12(1):1-14. doi:10.1186/s13287-021-02248-2
• Mobarak H, Heidarpour M, Rahbarghazi R, Nouri M, Mahdipour M. Amniotic fluid-derived exosomes improved spermatogenesis in a rat model of azoospermia. Life Sci. 2021;274:119336. doi:10.1016/j.lfs.2021.119336
• Zhuang X, Xiang X, Grizzle W, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol Ther. 2011;19(10):1769-1779. doi:10.1038/mt.2011.164
• Lässer C, Eldh M, Lötvall J. Isolation and characterization of RNA-containing exosomes. J Vis Exp. 2012;(59):1-6. doi:10.3791/3037
• Subedi P, Schneider M, Philipp J, et al. Comparison of methods to isolate proteins from extracellular vesicles for mass spectrometry-based proteomic analyses. Anal Biochem. 2019;584:113390. doi:10.1016/j.ab.2019.113390
• Hernandez-Valladares M, Aasebø E, Mjaavatten O, et al. Reliable FASP-based procedures for optimal quantitative proteomic and phosphoproteomic analysis on samples from acute myeloid leukemia patients. Biol Proced Online. 2016;18:13. doi:10.1186/s12575-016-0043-0
• Mutschelknaus L, Azimzadeh O, Heider T, et al. Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells. Sci Rep. 2017;7(1):12423. doi:10.1038/s41598-017-12403-6
• Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther. 2018;9(1):63. doi:10.1186/S13287-018-0791-7
• Casado-Díaz A, Quesada-Gómez JM, Dorado G. Extracellular vesicles derived from mesenchymal stem cells (MSC) in regenerative medicine: applications in skin wound healing. Front Bioeng Biotechnol. 2020;8:146. doi:10.3389/fbioe.2020.00146
• Noronha NC, Mizukami A, Caliári-Oliveira C, et al.Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies. Stem Cell Res Ther. 2019 May 2;10(1):131. doi:10.1186/s13287-019-1224-y.
• Joo HS, Suh JH, Lee HJ, Bang ES, Lee JM. Current Knowledge and Future Perspectives on Mesenchymal Stem Cell-Derived Exosomes as a New Therapeutic Agent. International Journal of Molecular Sciences. 2020; 21(3):727. doi:10.3390/ijms21030727
• Haraszti RA, Miller R, Dubuke ML, et al. Serum Deprivation of Mesenchymal Stem Cells Improves Exosome Activity and Alters Lipid and Protein Composition. iScience. 2019 Jun 28;16:230-241. doi:10.1016/j.isci.2019.05.029.