Artikel
Immune regulatory and stem cell functions of human bone marrow mesenchymal stromal cells exposed to hematoma factors in vitro
Suche in Medline nach
Autoren
Veröffentlicht: | 22. Oktober 2019 |
---|
Gliederung
Text
Objectives: The bone healing process involves inflammatory hematoma formation where the functional state of bone marrow mesenchymal stromal cells (MSC) governs the regenerative phase, final bone tissue restoration or disease progression. Although found as important, in the context of bone healing, communication of bone marrow MSC with immune cells is still not fully understood. Our aim was to evaluate immunomodulatory roles displayed by MSC pre-exposed to hematoma-mimicking hydrogels containing platelet rich plasma (PRP) or fibrin (FBR).
Methods: MSC were isolated from bone marrow of patients after informed consent and used up to 4th passage, while PRP was collected from pooled apheresis thrombocyte concentrates with 10-fold increased thrombocyte content. MSC were cultured in hydrogels containing 100% (PRP100%) or 50% (PRP50%) PRP, FBR (5mg/mL) or in monolayer for 72 hours. Afterwards, MSC were co-cultured with peripheral blood mononuclear cells (PBMC, pooled, n=5) in direct contact for 72 hours (corresponding to inflammatory hematoma phase). To test stem cell related properties of MSC released from hydrogels, proliferation, clonogenicity, surface marker expression and adherence were estimated. After co-cultures, proliferation, cell cycle, phenotype and intracellular interferon (IFN)-gamma production in PBMC were investigated. Clonogenicity was determined by colony forming unit-fibroblast assay, adherence by fluorometric detection of metabolic activity, while multicolor flow cytometry was used to assess cell proliferation, cycle progression and surface and intracellular protein expression.
Results and conclusion: Although PRP100%, PRP50% and FBR hydrogel conditions experienced by MSC did not changed their proliferation, their co-expression of MSC markers (CD90, CD44, CD73, CD105, CD13), clonogenic and adhesion capacity was reduced after pre-exposure to PRP100%. On the other side, PRP100% stimulated CD146 and CD31 marker expression in MSC. In MSC-PBMC co-cultures, pre-exposure of MSC with PRP100% reduced the proliferation rate and frequency of CD3+CD4+CD25+ lymphocytes and altered their distribution in S and G2/M cell cycle phase. In addition to sustaining lymphocytes in resting state, PRP100% exposed MSC (n=5) favored persistence of FOXP3+ lymphocytes within CD3+CD4+CD25+ PBMC (32±4.7% compared to 19±3.3% in co-cultures with control monolayer MSC, n=6). PRP100% exposed MSC reduced percentage of IFN-gamma producing CD3+ lymphocytes (from 1.22±0.3% to 0.3±0.08%, n=3). These results indicate that exposure to hematoma factors (predominantly PRP100%, while PRP50% and FBR had only moderate effects) might support the immune suppressive role, while simultaneously coinciding with truncated stem cell related properties of MSC. Better understanding of bone marrow MSC behavior during their encounter with immune cells in inflammatory milieu of fracture hematoma may point to their therapeutic or diagnostic significance in bone injuries.