The Immunological Profile of Adipose Mesenchymal Stromal/Stem Cells after Cell Expansion and Inflammatory Priming

Abstract

Background: AT-MSCs display great immunoregulatory features, making them potential candidates for cell-based therapy. This study aimed to evaluate the "RBC lysis buffer" isolation protocol and immunological profiling of the so-obtained AT-MSCs.

Methods: We established an immune-comparative screening of AT-MSCs throughout in vitro cell expansion (PM, P1, P2, P3, P4) and inflammatory priming regarding the expression of 28 cell-surface markers, 6 cytokines/chemokines, and 10 TLR patterns.

Findings: AT-MSCs were highly expandable and sensitive to microenvironment challenges, hereby showing plasticity in distinct expression profiles. Both cell expansion and inflammation differentially modulated the expression profile of CD34, HLA-DR, CD40, CD62L, CD200 and CD155, CD252, CD54, CD58, CD106, CD274 and CD112. Inflammation resulted in a significant increase in the expression of the cytokines IL-6, IL-8, IL-1β, IL-1Ra, CCL5, and TNFα. Depending on the culture conditions, the expression of the TLR pattern was distinctively altered with TLR1-4, TLR7, and TLR10 being increased, whereas TLR6 was downregulated. Protein network and functional enrichment analysis showed that several trophic and immune responses are likely linked to these immunological changes.

Conclusions: AT-MSCs may sense and actively respond to tissue challenges by modulating distinct and specific pathways to create an appropriate immuno-reparative environment. These mechanisms need to be further characterized to identify and assess a molecular target that can enhance or impede the therapeutic ability of AT-MSCs, which therefore will help improve the quality, safety, and efficacy of the therapeutic strategy.

Keywords: Toll-like receptors; adipose tissue; cell passaging; cell therapy; cytokines; immune biology; inflammation; mesenchymal stromal/stem cells; red blood cell lysis buffer; regenerative medicine.

Figure 1

The image is representative of the morphology of AT-MSCs obtained by the RBC lysis buffer protocol and cultured by the classical adherent method. The cells are visualized by light microscopy (scale bar = 500 µm), showing a fibroblast-like cell population.

Figure 2

Expression of endothelial/stromal markers in AT-MSCs exposed to inflammatory inducers. The flow cytometry values are expressed as the percentage (%) of positive cells and mean fluorescence intensity (MFI). The values are presented as mean ± SD and originate from six AT-MSC donors. ^## Significantly lower percentage or amount per cell in non-stimulated AT-MSCs compared to the consecutive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs compared to the consecutive passage (p-value < 0.05). ** Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs compared to regular AT-MSCs (p-value < 0.05).

Figure 3

Expression of human leukocyte antigen markers in AT-MSCs exposed to inflammatory inducers. The flow cytometry values are expressed as the percentage (%) of positive cells and mean fluorescence intensity (MFI). The values are presented as mean ± SD and originate from six AT-MSC donors. * Significantly higher percentage or amount per cell in inflammation-stimulated AT-MSCs compared to regular AT-MSCs (p-value < 0.05). ^## Significantly lower percentage or amount per cell in regular AT-MSCs compared to the consecutive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs compared to the consecutive passage (p-value < 0.05).

Figure 4

Expression of co-stimulatory molecules in AT-MSCs exposed to inflammatory inducers. The flow cytometry values are expressed as the percentage (%) of positive cells and mean fluorescence intensity (MFI). The values are presented as mean ± SD and originate from six AT-MSC donors. * Significantly higher percentage or amount per cell in inflammation-stimulated AT-MSCs compared to normal AT-MSCs (p-value < 0.05). ^## Significantly lower percentage or amount per cell in regular AT-MSCs compared to the successive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs compared to the successive passage (p-value < 0.05).

Figure 5

Expression of cell adhesion molecules in AT-MSCs exposed to inflammatory inducers. The flow cytometry values are expressed as the percentage (%) of positive cells and mean fluorescence intensity (MFI). The values are shown as mean ± SD and originate from six distinctive AT-MSC donors. * Significantly higher percentage or amount per cell in inflammation-stimulated AT-MSCs compared to regular AT-MSCs (p-value < 0.05). ^## Significantly lower percentage or amount per cell in regular AT-MSCs compared to the consecutive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs compared to the consecutive passage (p-value < 0.05).

Figure 6

Expression of immunoregulatory molecules in AT-MSCs exposed to inflammatory inducers. Flow cytometric analysis was utilized for the determination of the percentage (%) of positive cells and the protein expression per cell (MFI). The data are expressed as mean ± SD and originate from six AT-MSC donors. * Significantly higher percentage or amount per cell in inflammation-stimulated AT-MSCs compared to regular AT-MSCs (p-value < 0.05). ^## Significantly lower percentage or amount per cell in regular AT-MSCs compared to the successive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammatory primed AT-MSCs compared to the consecutive passage (p-value < 0.05).

Figure 7

Expression of natural killer ligands in AT-MSCs exposed to inflammatory inducers. Flow cytometric analysis was used to calculate the percentage of positive cells and the protein expression per cell (MFI). The data are presented as mean ± SD and originate from six AT-MSC donors. * Significantly higher percentage or amount per cell in inflammation-stimulated AT-MSCs versus regular AT-MSCs (p-value < 0.05). ^## Significantly lower percentage or amount per cell in regular AT-MSCs versus the consecutive passage (p-value < 0.05). ^$$ Significantly lower percentage or amount per cell in inflammation-stimulated AT-MSCs versus the consecutive passage (p-value < 0.05).

Figure 8

Cytokine and chemokine expression is upregulated in AT-MSCs after inflammatory stimulation. The gene expression levels are determined by qPCR analysis of untriggered and inflamed MSCs (INFL) during the primo culture (PM), early (EARLY), and late (LATE) passage (P). The values are expressed as mean ± SEM compared to the expression of the housekeeping gene (GAPDH). *, **, ***, **** Significant increase in expression of inflamed MSCs (INFL) versus regular MSCs (p-value: p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, p ≤ 0.0001, respectively).

Figure 9

Toll-like receptors (TLRs) are differentially expressed in AT-MSCs after inflammatory stimulation. The gene expression levels are determined by qPCR analysis of untriggered MSCs and inflamed MSCs (INFL) during the primo culture (PM), early (EARLY), and late (LATE) passage (P). The values are expressed as mean ± SEM compared to the expression of the housekeeping gene (GAPDH). *, **, ***, **** Significant increase in expression of inflamed MSCs (INFL) versus basic MSCs (p-value: p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, p ≤ 0.0001, respectively). ^$ Significant decrease in expression of inflamed MSCs (INFL) versus basic MSCs (p-value: p ≤ 0.05). ^§ Significantly higher expression compared to the following passage (p-value: p ≤ 0.05). ^# Significantly lower expression versus the consecutive passage (p-value: p ≤ 0.05).

Figure 10

Analysis of known and predicted immunoregulatory molecules, co-stimulatory molecules, cell adhesion molecules, cytokine, and TLR protein interactions using the STRING database. The interactions comprise direct (physical) and indirect (functional) associations. Network nodes represent proteins and edges signify protein–protein associations (specific and meaningful). The lines are considered as the existence of several types of evidence applied in predicting the associations (high confidence score 0.9). The interactions are shown in different colors: green is neighborhood evidence, blue is co-occurrence evidence, purple is experimental evidence, light blue is database evidence and black is co-expression evidence.