Title : Mesenchymal stromal cells as therapeutic tools in immune disorders: Promising evidence from in vitro results
Abstract:
Background: Mesenchymal Stromal Cells (MSCs), a multipotent stem cell population which can efficiently be obtained from different tissue sources, exert key specific regenerative and immunomodulatory properties. However, MSCs require priming through binding of IFN-γ to the receptor IGFR1, for the induction of their immunomodulatory properties. Moreover, the mediated immunomodulation by MSCs can be exerted either through direct contact (cell-cell interaction) or the paracrine production of anti-inflammatory biomolecules. Due to the great tolerabily and low immunogenecity that MSCs exhibited when allogeneically administrated, these stem cells are currently used in a great number of clinical trials (> 1300 studies, worldwide), including mostly their application against autoimmune diseases, graft versus host disease (GvHD) and cancer. In addition, clinical grade MSCs were evaluated for the efficient regulation of acute immune responses and cytokine release syndrome (CRS) in COVID-19 patients. Besides their beneficial effects, MSCs share different regenerative and immunoregulatory potential, among the different tissue origins, which may hamper their immunomodulatory properties.
Aim: The aim of this study was the evaluation of the functional properties of MSCs obtained either from fetal and adult origin, to possibly desipher their immunomodulatory potential.
Methods: MSCs derived from the human Wharton's Jelly (WJ, n=20) tissue and bone marrow (BM, n=20) were isolated, cryopreserved, expanded, and characterized according to the criteria outlined by the International Society for Cell and Gene Therapies (ISCT). WJ (n=20) and BM-MSCs (n=20) were stimulated with a culture medium containing IFN-γ (50 ng/ μl), 1% penicillin-streptomycin, and 1% L-glutamine for 48 h. The quantification of IL-1Ra, IL-6, IL-10, IL-13, TGF-β1, VEGF-a, FGF, PDGF, and IDO was performed using commercial ELISA kits. The expression of HLA-G1, G5, and G7 was also evaluated in WJ and BM-MSCs. WJ and BM-MSCs were co-cultured in 96 transwell plates with M0 macrophages, to evaluate their immunoregulatory properties, through the polarization effect. The determination of the HLA alleles of the MSCs was performed using the Next Generation Sequencing (HLA Holotype 11 loci, Omixon Inc., MiSeq, Illumina). The frequencies of the HLA alleles were estimated using the machine learning algorithms in R language.
Results: Thawed WJ and BM-MSCs exhibited a spindle-shaped morphology, successfully differentiated to "osteocytes", "adipocytes", and "chondrocytes", and in flow cytometric analysis were characterized by positivity for CD73, CD90, and CD105 (> 95%) and negativity for CD34, CD45, and HLA-DR (< 2%). Moreover, stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation, in comparison to unstimulated cells. The HLA-G isoforms (G1, G5, and G7) were successfully expressed by the unstimulated and stimulated WJ-MSCs. On the other hand, only weak expression of HLA-G1 was identified in BM-MSCs. Stimulated MSCs secreted high levels of IL-1Ra, IL-6, IL-10, IL-13, TGF-β1, FGF, VEGF, PDGF, and IDO in comparison to unstimulated cells (P < 0.05) after 12 and 24 h.Importanty, MSCs from both sources achieved to induce the M2 macrophage polarization, under in vitro conditions. The most frequent HLA alleles were determined, to identify potential association with the MSCs? immunomodulatory properties.
Conclusion: The results of this study showed that WJ-MSCs have greater immunoregulatory potential in terms of paracrine factors secretion, compared to the BM-MSCs. Taking into consideration the data presented herein, well-defined WJ-MSCs could be used as advanced therapeutic medicinal products (ATMPs) and alternative treatment option for severe immune disorders. In this way, a stem cell bank with MSCs lines could be established, in order to serve better the purposes of the precision medicine.
