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. 2019 Dec 2;9(1):18078.
doi: 10.1038/s41598-019-54462-x.

Thawed Mesenchymal Stem Cell Product Shows Comparable Immunomodulatory Potency to Cultured Cells In Vitro and in Polymicrobial Septic Animals

Yuan Tan  1 , Mahmoud Salkhordeh  1 , Jia-Pey Wang  1 , Andrea McRae  1 , Luciana Souza-Moreira  1 , Lauralyn McIntyre  2   3 , Duncan J Stewart  1   3 , Shirley H J Mei  4
Affiliations

Thawed Mesenchymal Stem Cell Product Shows Comparable Immunomodulatory Potency to Cultured Cells In Vitro and in Polymicrobial Septic Animals

Yuan Tan et al. Sci Rep. .

Abstract

Mesenchymal stem cells (MSCs) have been shown to exert immunomodulatory effects in both acute and chronic diseases. In acute inflammatory conditions like sepsis, cell therapy must be administered within hours of diagnosis, requiring "off-the-shelf" cryopreserved allogeneic cell products. However, their immunomodulatory potency, particularly in abilities to modulate innate immune cells, has not been well documented. Herein we compared the stabilities and functionalities of cultured versus thawed, donor-matched MSCs in modulating immune responses in vitro and in vivo. Cultured and thawed MSCs exhibited similar surface marker profiles and viabilities at 0 hr; however, thawed MSCs exhibited higher levels of apoptotic cells beyond 4 hrs. In vitro potency assays showed no significant difference between the abilities of both MSCs (donor-matched) to suppress proliferation of activated T cells, enhance phagocytosis of monocytes, and restore endothelial permeability after injury. Most importantly, in animals with polymicrobial sepsis, both MSCs significantly improved the phagocytic ability of peritoneal lavage cells, and reduced plasma levels of lactate and selected inflammatory cytokines without significant difference between groups. These results show comparable in vitro and in vivo immunomodulatory efficacy of thawed and fresh MSC products, providing further evidence for the utility of a cryopreserved MSC product for acute inflammatory diseases.

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Conflict of interest statement

The funding institution had no role in the conception, design or conduct of the study, data collection or analysis, interpretation or presentation of the data, or preparation, review or approval of the manuscript. We also like to declare the following conflicts of interest: D.J.S. holds a patent for MSC therapy for the treatment of acute lung injury, and S.H.J.M. has received personal fees from Northern Therapeutics that are outside of this submitted work. The remaining authors have disclosed that they do not have any conflicts of interest.

Figures

Figure 1
Figure 1
Assessment and comparison of cell viability of cultured and thawed MSCs over 6 hours. (A) Trypan blue exclusion method was used to assess viability (left panel) and calculate viable cell recoveries (right panel), with measurements taken at 0, 2, 4, and 6 hours. (B) Annexin V (AV) and Propidium Iodide (PI) staining of cells followed by flow cytometry analysis was used to assess populations of cells that were viable (left panel) and going through early (middle panel) or late (right panel) apoptosis, with measurements taken at 0, 2, 4, and 6 hours. (C) Representative flow cytometry data of AV/PI stained cells demonstrating percentage of late apoptotic cells (AV+/PI+, Q2); early apoptotic cells (AV+, Q3), and live cells (AV−/PI−, Q4). n = 3 independent experiments, with data represent mean ± SEM.
Figure 2
Figure 2
Surface marker profiles for cultured and thawed MSCs. Representative flow cytometric plots indicate positive markers: CD73, CD90, CD105; negative markers: CD14, CD19, CD34, CD45, and HLA-DR.
Figure 3
Figure 3
Cultured and thawed MSCs were comparably potent in inhibiting T cell proliferation. PBMCs were stained with carboxyfluorescein succinimidyl ester dye (CFSE), then activated with anti-CD3/CD28 Dyna-beads. CFSE-labeled PBMCs were co-cultured with MSCs for 5 days before analysis by flow cytometer. (A) Representative CFSE histograms of activated PBMCs without MSCs, activated PBMCs co-cultured with cultured MSCs, or activated PBMCs co-cultured with thawed MSCs. (B) Percent inhibition of activated PBMCs after co-culture with cultured or thawed MSCs. n = 3~6 experiments, with bars represent mean ± SEM (2-tailed unpaired t-test corrected with Holm-Sidak method). (C) Microscopic images of activated PBMC without MSCs (shown as aggregated colonies), or with different donor-derived MSCs after co-culture for 5 days. Scale bar = 100 µm.
Figure 4
Figure 4
Effect of cultured and thawed MSCs on PBMC’s phagocytic capacity. (A) Representative flow cytometric plots of naïve PBMCs, LPS-treated PBMCs without and with MSCs demonstrating the PBMC’s ability to phagocytose bacteria as indicated by the percentage of CD14+ cells positive for green fluorescent signal. (B) Naïve PBMCs were pre-treated with LPS for 18 hours and co-cultured with cultured and thawed MSCs from three different donors for 24 hours. PBMCs were harvested and incubated with fluorescent tagged E. coli and analysed by flow cytometry to show the percentage of CD14+/E. coli positive cells. n = 3~6 experiments, with bars representing mean ± SEM. Group comparisons were analyzed by one-way ANOVA with Bonferroni’s post hoc test. ****p < 0.0001, LPS-treated PBMC versus non-treated group. ns = not significant between groups compared.
Figure 5
Figure 5
Single cell imaging of PBMC’s capacity to engulf fluorescent bacteria. AMNIS Imaging flow cytometer digital images show representative CD14+ PBMC populations (purple) and the ability of cultured and thawed MSCs to rescue the PBMC’s capacity to engulf the pHrodo Bioparticles (green). The PBMC nuclei are stained with DRAQ5 in red. The morphology of the cells is shown by bright-field microscopy and the PBMC nuclei are stained with DRAQ5 in red. Scale bar = 10 µm.
Figure 6
Figure 6
Cultured and thawed MSCs were comparable in their abilities to recover endothelial cell permeability post-LPS injury. Endothelial cells (ECs) were seeded in a transwell and treated with LPS for 6 hours. Cells were then co-cultured for 24 hours with MSCs that were seeded in the lower receiver wells. After co-culture, FITC-dextran was added to the top of the transwells. Permeability of the EC monolayer was determined by taking samples from the lower compartment to measure FITC-dextran levels. Group comparisons were analyzed by one-way ANOVA with Bonferroni’s post hoc test. ****p < 0.0001, LPS-treated ECs versus non-treated group. ns = not significant between groups compared.
Figure 7
Figure 7
Cultured and thawed MSCs were comparable in improving bacterial clearance and systemic inflammation in a murine model of acute inflammatory injury (cecal-ligation-puncture model). (A) Peritoneal lavage cells were incubated with fluorescent tagged E. coli followed by flow cytometric analysis to assess the ability of the peritoneal cells to phagocytose bacteria. (B) Peritoneal lavage fluid was plated on blood agar to quantify the colony forming units (CFUs) and determine the peritoneal bacterial load. Plasma samples were evaluated to detect (C) lactate, and (D) cytokine levels of systemic inflammation. Group comparisons were analyzed by one-way ANOVA with Dunnett’s post hoc test. #p < 0.05, ###p < 0.001 and ####p < 0.0001, sham/vehicle versus CLP/vehicle group. *p < 0.05, **p < 0.01 and ****p < 0.0001, CLP/saline versus MSCs-treated group. ns = not significant between groups compared.
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