Extracellular Vesicles from Different Mesenchymal Stem Cell Types Exhibit Distinctive Surface Protein Profiling and Molecular Characteristics: A Comparative Analysis

Abstract

The current medical need to respond to different diseases has sparked great interest in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) due to their great regenerative potential and as drug carriers by playing a critical role in cell-cell communication. However, due to their heterogeneity, there is no standardized universal method for their identification and characterization, which limits their clinical application. This study, following the recommendations and methodologies proposed by MISEV2023 for the characterization of EVs, shows for the first time a detailed morphological, protein, and biochemical comparison between EVs derived from three different MSCs sources (placenta, endometrium, and dental pulp). The information obtained from the different applied assays suggests that there are substantial differences between one EVs source and another. It also offers valuable insights that provide the guidelines to ease their profiling and therefore improve their selection, in order to speed up their use and clinical application; additionally, the knowledge obtained from each characterization test could facilitate new researchers in the field to choose a specific cell source to obtain EVs and select the appropriate methods that provide the necessary information according to their requirements.

Keywords: EVs of human dental pulp-derived mesenchymal stem cells (hDP-MSC-EVs); EVs of human endometrium-derived mesenchymal stem cells (hE-MSC-EVs); EVs of human placenta-derived mesenchymal stem cells (hP-MSC-EVs); extracellular vesicles (EVs).

Figure 1

Biogenesis and release of extracellular vesicles. Exosomes are formed by multivesicular bodies (MVBs). Ectosomes are formed by the plasma membrane, and apoptotic bodies by disassembly of an apoptotic cell or programmed cell death (drawn using the BioRender.com).

Figure 2

Electronic micrographs of extracellular vesicles derived from mesenchymal stem cells (MSC-EVs). (A) Morphological assessment of MSC-EVs at the single-vesicle level by transmission electron microscopy; scale bar: 50 nm. (B) Presence and morphological assessment of MSC-EVs by scanning electron microscopy; scale bar: 2 μm (EVs are marked with red arrows to facilitate their observation). Abbreviations: hE-MSC-EVs, EVs of human endometrium-derived mesenchymal stem cells; hP-MSC-EVs, EVs of human placenta-derived mesenchymal stem cells; hDP-MSC-EVs, EVs of human dental pulp-derived mesenchymal stem cells.

Figure 3

Dynamic light scattering. (A) Representative size distribution profile assessment of MSC-EVs obtained by the average diameter and the standard deviation of each sample. (B) The average polydispersity index (PDI) of MSC-EVs, and the standard deviation of each sample. (C) The zeta potential measurement of MSC-EVs, the average of the net negative charge, and the standard deviation of each sample. Note: the measures of size distribution, PDI, and zeta potential were calculated by the consecutive run of three representative samples. Abbreviations: hP-MSC-EVs, EVs of human placenta-derived mesenchymal stem cells; hE-MSC-EVs, EVs of human endometrium-derived mesenchymal stem cells; hDP-MSC-EVs, EVs of human dental pulp-derived mesenchymal stem cells.

Figure 4

Surface marker profile of extracellular vesicles (EVs) isolated from three different sources of mesenchymal stem cells (MSCs) (dental pulp, placenta, and endometrium), profiled with a multiplex bead-based flow cytometry assay for the detection of EV surface signatures. (A) Results after analyzing the extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) compared to the medium control (no MSC-EVs), identifying 39 distinct bead populations based on their fluorescence in the FITC vs. PE channels. Dot plots were used to visualize the APC-stained bead populations. Calibration beads (gate selection by size and granularity), first column. The determination of the target area and the 39 epitope areas is given in the second column, and the third column shows the adherence of MSC-EVs to the beads. (B) Graphical representation of the median APC fluorescence intensity detected (background subtracted for each marker analyzed by the MACSPlex EV IO kit for all MSC-EV samples). Abbreviations: hP-MSC-EVs, EVs of human placenta-derived mesenchymal stem cells; hE-MSC-EVs, EVs of human endometrium-derived mesenchymal stem cells; hDP-MSC-EVs, EVs of human dental pulp-derived mesenchymal stem cells.

Figure 5

Average FTIR spectra of EVs obtained from three different MSC sources (hP-MSC-EVs, hE-MSC-EVs and hDP-MSC-EVs). (A) FTIR spectrum after application of the second derivative, showing the presence of the secondary protein structures, phosphates, carbohydrates, and lipids. (B) FTIR spectrum without modification, showing the presence of amides. Abbreviations: hP-MSC-EVs, EVs of human placenta-derived mesenchymal stem cells; hE-MSC-EVs, EVs of human endometrium-derived mesenchymal stem cells; hDP-MSC-EVs, EVs of human dental pulp-derived mesenchymal stem cells.

Figure 6

Second derivative of the EVs’ Raman fingerprints in the spectral range (A) 700 cm⁻¹ to 1700 cm⁻¹ (left) and (B) 2850 cm⁻¹ to 3200 cm⁻¹ (right) of hP-MSC-EVs, hE-MSC-EVs, and hDP-MSC-EVs. Average Raman spectra obtained using an excitation wavelength of 785 nm and 25 s for 15 pulsations of exposure for each spectrum. Abbreviations: hP-MSC-EVs, EVs of human placenta-derived mesenchymal stem cells; hE-MSC-EVs, EVs of human endometrium-derived mesenchymal stem cells; hDP-MSC-EVs, EVs of human dental pulp-derived mesenchymal stem cells.

Figure 7

Isolation and identification of hP-MSCs, hE-MSCs and hDP-MSCs. (A) Markers (CD44, CD73, CD90, and CD105) detected by flow cytometry. (B) Micrography of adherent MSCs observed under a microscope. (C) Multipotency capacity of MSCs evaluated by Oil Red O staining.

Figure 8

Experimental overview of the short tube protocol (drawn using the BioRender.com).