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

FIGURE 1

Multiplex bead‐based EV flow cytometry assay principle and example data output. (a) Principle and workflow of multiplex bead‐based EV flow cytometry assay platform. (b) Example surface marker profiles for HEK293T‐ and MSC‐derived EVs for the previously established immuno‐oncology panel. (c) Flow cytometric gating strategy for the MSC assay measured in two separate panels. Single capture beads were gated based on FSC‐A versus SSC‐A parameters, and bead subpopulations were identified by green (525/50 nm filter) versus orange light (585/40 nm filter) emitted after excitation with a blue laser (488 nm). Signals derived from APC‐conjugated detection antibodies were quantified in the R1‐A channel (655–730 nm filter) after excitation with a red laser (640 nm). (d) Example dataset showing differential surface marker profiles for HEK293T‐ and MSC‐derived EVs.

FIGURE 2

Inter‐laboratory assay assessment. (a) Schematics summarising the study. MSC‐EVs were prepared and measured by multiplex‐bead based EV flow cytometry assays in participating labs before data was normalised and analysed centrally. (b) Example data showing assay analysis including gating strategy and capture bead identification steps for all lab participants (note that EVs provided by Lab 4 were measured in Lab 3). See Section 2, Table 1 and Table S1 for instrument and assay details.

FIGURE 3

Normalised protein surface marker data from all labs. Clustering heatmap combining the normalised signals of all samples for all markers. Add., media additive; AT, adipose tissue; BM, bone marrow; CB, umbilical cord blood; ESC, embryonic stem cell; hPL, human platelet lysate; WJ, Wharton Jelly.

FIGURE 4

Candidate MSC‐EV marker selection and characterisation. (a) Summary of selected MSC‐MBFCM surface markers classified as either consistently positive or negative on all MSC‐EV samples included in this study. (b) Characterisation of MSC‐EV markers classified as positive on all MSC‐EV samples by using single anti‐tetraspanin antibodies for detection. Add., media additive; AT, adipose tissue; BM, bone marrow; CB, umbilical cord blood; ESC, embryonic stem cell; hPL, human platelet lysate; WJ, Wharton Jelly.

FIGURE 5

Comparison of MSC and MSC‐EV surface markers. ^*Surface markers in ISCT minimal criteria for defining MSC (Dominici et al., 2006). ^#MSC‐EV surface marker detected by proteomics (van Balkom et al., 2019). ^**Detected as positive on MSC‐EVs from MSCs cultured in hPL and 5 other MSC EV preparations. ^***Detected as positive on MSC‐EVs from MSCs cultured in hPL only. Note: ‘general EV marker’ refers to more common presence also on EVs from other sources, not necessarily from all sources.