Abstract
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.
Highlights
Mesenchymal stromal cells (MSCs), multipotent cells that can be isolated from a wide range of fetal, perinatal, and adult tissues, have emerged as a promising cell type for regenerative medicine [1, 2]
Since the maintenance culture medium was supplemented with 10% fetal bovine serum (FBS), we analyzed the levels of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) in fresh cell-free maintenance medium, to exclude the possibility that we were detecting VEGF and MCP-1 from the FBS. Both VEGF and MCP-1 levels were below the detection limit of the assay, which was validated for the detection of rat proteins. These results showed that the physiological release of IL-6, MCP-1, and VEGF by cultured rBM-MSCs can be enhanced by a brief exposure to LPS, whereas the production of other proinflammatory (IFN-γ, IL-1β, IL-2, IL-18, and tumor necrosis factor- (TNF-)α) and anti-inflammatory cytokines (IL-4, IL-10, IL13) was not observed in any conditions
Considering that VEGF, IL-6, and MCP-1 are involved in peripheral nerve repair and regeneration [42, 43, 47], we evaluated the proregenerative capacity of rBM-MSCs and LPSpreconditioned rBM-MSCs in a rat model of sciatic nerve transection
Summary
Mesenchymal stromal cells (MSCs), multipotent cells that can be isolated from a wide range of fetal, perinatal, and adult tissues, have emerged as a promising cell type for regenerative medicine [1, 2]. Human, equine, and murine MSCs obtained from different tissues have been shown to express functional Toll-like receptors (TLR) [12,13,14,15,16]. MSCs acquire a distinctive functional phenotype when stimulated with polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of double-stranded RNA that activates TLR3 [13, 19, 20]. These observations have led to the use of TLR agonists in preconditioning protocols aimed at boosting and/or modifying the therapeutic effects of MSCs [21,22,23]
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