Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures

Vivian V.T. Nguyen; Joshua A. Welsh; Tobias Tertel; Andre Choo; Simonides I. van de Wakker; Kyra A.Y. Defourny; Bernd Giebel; Pieter Vader; Jayanthi Padmanabhan; Sai Kiang Lim; Esther N.M. Nolte-'t Hoen; Marianne C. Verhaar; R. Beklem Bostancioglu; Antje M. Zickler; Jia Mei Hong; Jennifer C. Jones; Samir EL Andaloussi; Bas W.M. van Balkom; André Görgens

doi:10.1002/jev2.12463

Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures

Vivian V.T. Nguyen, Joshua A. Welsh, Tobias Tertel, Andre Choo, Simonides I. van de Wakker, Kyra A.Y. Defourny, Bernd Giebel, Pieter Vader, Jayanthi Padmanabhan, Sai Kiang Lim, Esther N.M. Nolte-'t Hoen, Marianne C. Verhaar, R. Beklem Bostancioglu, Antje M. Zickler, Jia Mei Hong, Jennifer C. Jones, Samir EL Andaloussi, Bas W.M. van Balkom^*, André Görgens^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Original language	English
Article number	e12463
Number of pages	18
Journal	Journal of Extracellular Vesicles
Volume	13
Issue number	6
DOIs	https://doi.org/10.1002/jev2.12463
Publication status	Published - Jun 2024

Bibliographical note

Publisher Copyright:
© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

Funding

The authors thank Dr Stefan Wild and Ute Heider (both Miltenyi Biotec B.V & CO. KG) for providing reagents and critical reading of the manuscript. This research is supported by the Industry Alignment Fund Pre-positioning Programme Human Health and Potential domain (IAF-PP HHP domain; H19H6a0026) and administered by the Agency for Science, Technology and Research (A*STAR). The REDUCE MORE! Project was funded by Health similar to Holland TKI-LSH, grant number LSHM18045 (to B.W.M. v.B.). This research was also funded by the Dutch Heart Foundation CVON2014-11 'RECONNECT' and 'RECONNEXT' (to M.C.V.), the RECONNECT YTP 'CHIPS' grant, the Dutch Society for the Replacement of Animal Testing (Stichting Proefdiervrij), and the Utrecht University 3Rs stimulus fund project 'MOOI!', (to B.W.M.v.B.) grants; the EU H2020 research and innovation programme under Marie S. Curie Cofund RESCUE grant agreement No 801540 (to M.C.V.) and the Gravitation Program 'Materials Driven Regeneration', funded by the Netherlands Organization for Scientific Research (024.003.013) (to M.C.V.). The authors acknowledge the support of the partners of 'Regenerative Medicine Crossing Borders' (RegMed XB), Powered by Health similar to Holland, Top Sector Life Sciences & Health (to M.C.V) and the Van Herk Foundation (S.I.v.d.W). A.G. is an International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholar 2019-2024 and is supported by a Karolinska Institutet Network Medicine Global Alliance grant.

Funders	Funder number
RECONNECT YTP 'CHIPS' grant
Miltenyi Biotec B.V
Industry Alignment Fund Pre-positioning Programme Human Health and Potential domain	H19H6a0026
Agency for Science, Technology and Research (A*STAR) - Healthsimilar toHolland TKI-LSH	LSHM18045
Dutch Heart Foundation	CVON2014-11
Dutch Society for the Replacement of Animal Testing
Utrecht University 3Rs stimulus fund project 'MOOI!'
EU H2020 research and innovation programme under Marie S. Curie Cofund RESCUE	801540
Netherlands Organization for Scientific Research	024.003.013
Van Herk Foundation (S.I.v.d.W)
International Society for Advancement of Cytometry (ISAC)	2019-2024
Karolinska Institutet Network Medicine Global Alliance grant

Keywords

culture conditions
exosomes
extracellular vesicles
inter-laboratory assessment
mesenchymal stromal cells
MSC
MSC-marker
quality control
regenerative medicine
standardisation
surface markers

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J of Extracellular Vesicle - 2024 - Nguyen - Inter‐laboratory multiplex bead‐based surface protein profiling of MSC‐derivedFinal published version, 6.52 MBLicence: CC BY-NC

Cite this

Nguyen, V. V. T., Welsh, J. A., Tertel, T., Choo, A., van de Wakker, S. I., Defourny, K. A. Y., Giebel, B., Vader, P., Padmanabhan, J., Lim, S. K., Nolte-'t Hoen, E. N. M., Verhaar, M. C., Bostancioglu, R. B., Zickler, A. M., Hong, J. M., Jones, J. C., EL Andaloussi, S., van Balkom, B. W. M., & Görgens, A. (2024). Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures. Journal of Extracellular Vesicles, 13(6), Article e12463. https://doi.org/10.1002/jev2.12463

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title = "Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures",

abstract = "Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.",

keywords = "culture conditions, exosomes, extracellular vesicles, inter-laboratory assessment, mesenchymal stromal cells, MSC, MSC-marker, quality control, regenerative medicine, standardisation, surface markers",

author = "Nguyen, {Vivian V.T.} and Welsh, {Joshua A.} and Tobias Tertel and Andre Choo and {van de Wakker}, {Simonides I.} and Defourny, {Kyra A.Y.} and Bernd Giebel and Pieter Vader and Jayanthi Padmanabhan and Lim, {Sai Kiang} and {Nolte-'t Hoen}, {Esther N.M.} and Verhaar, {Marianne C.} and Bostancioglu, {R. Beklem} and Zickler, {Antje M.} and Hong, {Jia Mei} and Jones, {Jennifer C.} and {EL Andaloussi}, Samir and {van Balkom}, {Bas W.M.} and Andr{\'e} G{\"o}rgens",

note = "Publisher Copyright: {\textcopyright} 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.",

year = "2024",

month = jun,

doi = "10.1002/jev2.12463",

language = "English",

volume = "13",

journal = "Journal of Extracellular Vesicles",

issn = "2001-3078",

publisher = "John Wiley & Sons Inc.",

number = "6",

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Nguyen, VVT, Welsh, JA, Tertel, T, Choo, A, van de Wakker, SI, Defourny, KAY, Giebel, B, Vader, P, Padmanabhan, J, Lim, SK, Nolte-'t Hoen, ENM, Verhaar, MC, Bostancioglu, RB, Zickler, AM, Hong, JM, Jones, JC, EL Andaloussi, S, van Balkom, BWM & Görgens, A 2024, 'Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures', Journal of Extracellular Vesicles, vol. 13, no. 6, e12463. https://doi.org/10.1002/jev2.12463

TY - JOUR

T1 - Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures

AU - Nguyen, Vivian V.T.

AU - Welsh, Joshua A.

AU - Tertel, Tobias

AU - Choo, Andre

AU - van de Wakker, Simonides I.

AU - Defourny, Kyra A.Y.

AU - Giebel, Bernd

AU - Vader, Pieter

AU - Padmanabhan, Jayanthi

AU - Lim, Sai Kiang

AU - Nolte-'t Hoen, Esther N.M.

AU - Verhaar, Marianne C.

AU - Bostancioglu, R. Beklem

AU - Zickler, Antje M.

AU - Hong, Jia Mei

AU - Jones, Jennifer C.

AU - EL Andaloussi, Samir

AU - van Balkom, Bas W.M.

AU - Görgens, André

PY - 2024/6

Y1 - 2024/6

N2 - Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

AB - Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

KW - culture conditions

KW - exosomes

KW - extracellular vesicles

KW - inter-laboratory assessment

KW - mesenchymal stromal cells

KW - MSC

KW - MSC-marker

KW - quality control

KW - regenerative medicine

KW - standardisation

KW - surface markers

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U2 - 10.1002/jev2.12463

DO - 10.1002/jev2.12463

M3 - Article

AN - SCOPUS:85196092247

SN - 2001-3078

VL - 13

JO - Journal of Extracellular Vesicles

JF - Journal of Extracellular Vesicles

IS - 6

M1 - e12463

ER -

Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures

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Bibliographical note

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Keywords

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