Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Abstract

Background: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation.

Methods: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform.

Results: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege.

Conclusions: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Keywords: Apoptosis; Bioenergetics; Immunoprivilege; Lipidomics; Mesenchymal stem cells; Passage; Proteomics.

Fig. 1

Assessment of doubling time and immunoprivilege of MSCs. a Population doubling of MSCs at different passages was determined using trypan blue cell viability assay. The cells were plated in equal numbers followed by calculating the live cell number after 96 h of culture. There was no significant difference found in population doubling time of cells at different passages. b, c MSCs were cocultured with leukocytes (with or without E06 blocking antibody) for 72 h at a ratio of 1:10 (MSCs:leukocytes). b Leukocyte-mediated cytotoxicity in MSCs at different passages was determined by cytotoxicity assay kit using flow cytometry. There was no significant difference found in the level of cytotoxicity at different passages in the presence of leukocytes alone or in the presence of leukocytes and E06 antibody. c Western blot analysis was performed to determine the levels of the pro- and antiapoptotic proteins Bax and Bcl-xL. There was no significant difference observed in the Bax/Bcl-xl ratio in MSCs at different passages in the presence of leukocytes alone or in the presence of leukocytes and E06 antibody. Data are represented as mean ± SD (n = 3–6). ML, MSCs + leukocytes; MLA, MSCs + leukocytes + E06 antibody; MSC, mesenchymal stem cells

Fig. 2

Effect of MSCs on leukocyte proliferation and the secretion profile of leukocytes. Leukocytes were cocultured with MSCs at passages 3, 5, and 7 at a ratio of 1:10 (MSCs:leukocytes) for 72 h. a Leukocyte proliferation was measured by flow cytometry. The data are represented as different stages of the cell cycle: G0/G1 phase, S phase, and M phase. The extent of leukocyte proliferation by MSCs did not change with an increase in passage number since there was no significant difference found in the number of leukocytes at different stages of the cell cycle among different passages. The effect of an increase in passage number of MSCs on the secretion of b anti-inflammatory and c proinflammatory cytokines by leukocytes was analyzed using ELISA array. The results indicate that MSCs at different passages had no significant effect on the secretion profile of leukocytes. Data are represented as mean ± SD (n = 3–4). GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; LC, leukocytes alone; ML3, leukocytes cocultured with MSCs at P3; ML5, leukocytes cocultured with MSCs at P5; ML7, leukocytes cocultured with MSCs at P7; TNF, tumor necrosis factor

Fig. 3

Measurement of cellular bioenergetics using XF24 Seahorse analyzer. Mesenchymal stem cells (MSCs) at passages 3, 5, and 7 were cocultured with leukocytes (with or without E06 blocking antibody) at a ratio of 1:10 (MSCs:leukocytes) for 72 h. a Basal respiration rate, b spare respiratory capacity, c coupling efficiency, and d maximal respiration rate were measured in MSCs. There were no significant changes observed in these parameters in MSCs with the increase in passage number, in the presence of leukocytes, and in the presence of E06 antibody. The values are normalized to cell number in each well. Data are represented as mean ± SD (n = 4–6). MSC + L,  MSCs + leukocytes; MSC + L + Ab,  MSCs + leukocytes + E06 antibody

Fig. 4

Oxylipidome profile of MSCs by LC/MS was carried out at passages 3, 5, and 7. a One-way analysis of variance (ANOVA) plot with a p value threshold of 0.05. b Clustered heatmap (distance measure using euclidean, and clustering algorithm using ward) showing the intensity of 55 ox-PC compounds. Each row represents data for a specific ox-PC compound and each column represents an individual passage (P3, P5, and P7). All values are log-normalized values of detected abundance for each ox-PC compound. The colors changing from high (red) to low (blue) correspond to the different intensity level of ox-PCs (n = 3)

Fig. 5

Whole-cell proteome analysis of MSCs at passages 3 and 7 was performed using the LC/MS proteomic platform to determine the changes in different proteins with the increase in passage number. a Volcano plot of all the proteins shows no significant changes in all but 18 (shown in red color) proteins (p < 0.05). b–f The values of proteins involved in different pathways including cellular senescence (b), immunological synapse (c), glycolysis (d), tricarboxylic acid (TCA) cycle (e), and oxidative phosphorylation (f). Log2 fold change ratios of protein values at P7 versus P3 were calculated. The protein values between −1 and + 1 were considered to be normal and not changing significantly. Values higher than +1 indicate significant upregulation (at P7 compared to P3) of the protein and values lower than −1 indicate significant downregulation of the protein levels (n = 3)