Functional heterogeneity of mesenchymal stem cells and their therapeutic potential in the K18-hACE2 mouse model of SARS-CoV-2 infection

Abstract

Background: Despite many years of investigation into mesenchymal stem cells (MSCs) and their potential for treating inflammatory conditions such as COVID-19, clinical outcomes remain variable due to factors like donor variability, different tissue sources, and diversity within MSC populations. Variations in MSCs' secretory and proliferation profiles, and their proteomic and transcriptional characteristics significantly influence their therapeutic potency, highlighting the need for enhanced characterization methods to better predict their efficacy. This study aimed to evaluate the biological characteristics of MSCs from different tissue origins, selecting the most promising line for further validation in a K18-hACE2 mouse model of SARS-CoV-2 infection.

Methods: We studied nine MSC lines sourced from either bone marrow (hBMMSC), dental pulp (hDPMSC), or umbilical cord tissue (hUCMSC). The cells were assessed for their proliferative capacity, immunophenotype, trilineage differentiation, proteomic profile, and in vitro immunomodulatory potential by co-culture with activated lymphocytes. The most promising MSC line was selected for further experimental validation using the K18-hACE2 mouse model of SARS-CoV-2 infection.

Results: The analyzed cells met the minimum criteria for defining MSCs, including the expression of surface molecules and differentiation capacity, showing genetic stability and proliferative potential. Proteomic analysis revealed distinct protein profiles that correlate with the tissue origin of MSCs. The immunomodulatory response exhibited variability, lacking a discernible pattern associated with their origin. In co-culture assays with lymphocytes activated with anti-CD3/CD28 beads, all MSC lines demonstrated the ability to inhibit TNF-α, to induce TGF-β and Indoleamine 2,3-dioxygenase (IDO), with varying degrees of inhibition observed for IFN-γ and IL-6, or induction of IL-10 expression. A module of proteins was found to statistically correlate with the potency of IL-6 modulation, leading to the selection of one of the hUCMSCs as the most promising line. Administration of hUCMSC to SARS-CoV-2-infected K18 mice expressing hACE2 was effective in improving lung histology and modulating of a panel of cytokines.

Conclusions: Our study assessed MSCs derived from various tissues, uncovering significant variability in their characteristics and immunomodulatory capacities. Particularly, hUCMSCs demonstrated potential in mitigating lung pathology in a SARS-CoV-2 infection model, suggesting their promising therapeutic efficacy.

Keywords: COVID-19; Heterogeneity; Immunomodulation; K18-hACE2; Mesenchymal stem cells.

Fig. 1

MSC characterization (according to ISCT criteria). (A) MSC surface molecules were assessed by flow cytometry; (B) Representative images of MSC lines: control, grown in culture medium (bright field); exposed to adipogenic differentiation medium, Oil Red staining; chondrogenic differentiation medium, Alcian blue staining; and osteogenic differentiation medium, Alizarin red staining (20X magnification) lineages; (C) representative G-band karyotypes of 46, XY and 46, XX; (D) growth curve from MSC lines; (E) β-gal senescence-associated dosages from MSC lines

Fig. 2

Comparison of the proteomic profile and identification of differentially expressed proteins among the MSCs. (A) Venn diagram depicting the overlap in protein content among samples of hUCMSc, hDPMSCs, and hBMMSCs. (B) Hierarchical cluster heatmap of DEPs (n = 187) found in pairwise group comparisons (hUCMSc vs. hBMMSCs, hUCMSc vs. hDPMSCs and hDPMSCs vs. hBMMSCs). (C) The most relevant biological processes related to DEPs identified in each comparison

Fig. 3

Weighted correlation network analysis based on expression profiles identified in proteomic analysis. Heatmap with immunomodulatory potency measurements (IDO, kynurenines, TSG6, TGF-β, IFN-γ, IL-6, IL-10, and inhibition of lymphocyte proliferation), obtained from a functional assay - coculture of MSCs with activated PBMCs - normalized by z-score for hUCMSCs, hDPMSCs, and hBMMSCs. (A) WGCNA cluster dendrogram and module assignment (B). The branches indicate clusters of highly connected proteins. The colors on the horizontal bar represent the modules. Analysis of module-trait relationships (C). Each row corresponds to a ME, and each column corresponds to immunomodulation indicators. Each cell contains a corresponding correlation and P-value of modules with various immunomodulation indicators. Scatterplot of MM and GS from the red module and potency of inhibition of IL-6 (D), inhibition of IFNy (E), inhibition of TNF-α (F), inhibition of Lymphocyte proliferation (G), and induction of IL-10 (H). Protein hubs were highlighted in yellow. Gene Ontology for enriched proteins in the red module: Molecular process (I), Component Cellular (J), and biological process (K)

Fig. 4

Administration of hUCMSCs mitigates pathological damage in the lungs of K18-hACE mice inoculated with SARS-CoV-2. Left panels: Representative photomicrographs of lung parenchyma stained with hematoxylin-eosin. (A) in the CTRL group, lung architecture presents normal alveolar duct (D) and airways (AW), C: capillary; (B) in the SARS-CoV-2 group, lung damage was observed, with areas of alveolar collapse (asterisk), airway edema (ED), areas of hemorrhage (H), interstitial edema (arrows); (C) administration of MSCs decreased alveolar duct collapse (AD), airway inflammation (AW) and interstitial edema (arrows). Right panels: Cumulative diffuse alveolar damage (DAD) score (H) representing injury from hemorrhage (D), collapse (E), inflammation (F), and edema (G). Boxes show the interquartile range (P25–P75) range, whiskers denote the range (minimum-maximum), and horizontal lines represent the median of 5–6 animals per group. *Significantly different from the CTRL group (P < 0.05)

Fig. 5

Treatment with MSCs reduced the amplification of inflammation-associated cytokines and chemokines in lung tissue from SAR-CoV-2-infected mice. Levels of the proinflammatory cytokine (A) TNF-α, (B) 1L-1 α, C) IL-1-β, (D) IFN-g, (E) IL-5, (F) IL-2, (G) chemokines KC, and (H) IL-17a, (I) antiinflammatory cytokine IL-10, (J) Eotaxin, (K) Granulocyte colony-stimulating factor (G-CSF), and (L) interleukin-12-p40 were measured by multiplex immunoassay. Group comparisons were analyzed by unpaired Student’ t-test. Values are mean ± SD (*P < 0.05, **P < 0.01, and ***P < 0.001 SAR-CoV-2/saline versus MSC-treated group, n = 4–6 per group)

Fig. 6

Assessment of gene expression related to lung inflammation following hUCMSC treatment in SARS-CoV-2-infected mice. (A-E) Comparison of mRNA expression of TNF-α, IL-6, IL-1β, TGFB, Arg-1, IFN-y, IFN-A and IFN-B in the lung from SARS-CoV-2 mice with or without hUCMSC administration (n = 5–6/group). Data presented as mean ± SD of individuals included in each group. *P < 0.05, **P < 0.01 and # P < 0.05, significance levels for comparison between SARS-CoV-2-infected and MSC-treated groups