MSC therapy is considered a promising tool in regenerative medicine for repair of damaged tissues. It is believed that MSCs contribute significantly to wound healing and tissue regeneration by secretion of multiple tropic factors and ultimately differentiate into functional tissue-specific cells [40, 41]. Previously, we and others have reported the beneficial effects of MSC therapy in animal models of various human diseases such as rheumatoid arthritis [2, 40, 42], acute lung injury , kidney damage  and acute myocardial infarction . MSCs are also known for their immunomodulatory properties primarily mediated by cytokines or regulatory T cells [42, 46]. These multifunctional properties of MSCs indicate their potential in cell-based therapies and regenerative medicine.
Although increasing evidence suggests the beneficial effects and therapeutic use of MSCs for tissue regeneration and recovery, the major obstacle is failure in migration of large numbers of cells towards injured tissues . Various methods are reported for manipulation of MSCs to enhance or improve their recruitment and homing to damaged tissues. One popular approach is to modulate the natural adhesive machinery of MSCs that comprises chemokine receptors and adhesion molecules . Genetic manipulation of preexisting adhesion molecules, membrane modifications, treatment with cytokines and certain chemical factors enhance the expression of molecules required for cell migration [23, 25, 45, 47–49]. Although genetic manipulation of MSCs appears a promising tool to enhance their migration, it may have long-term, unforeseen effects on cellular function and may involve ethical issues and practical difficulties. Pretreatment with cytokines has led to some success in this area; for example, treatment with IFN-γ leads to increased CXCR4 expression and migration of MSCs towards the site of damage in mouse model of colitis . In a similar model, pretreatment with IL-1β is shown to increase migration of MSCs towards the inflammation through upregulation of CXCR4 . However, pretreatment of MSCs with these proinflammatory cytokines may lead to inflammation and cell death in nontargeted tissues and may raise several safety concerns . Also, it is reported that IFN-γ induces the expression of MHC-class II on MSCs, making them more immunogenic . Exposure of MSCs to hypoxia also leads to enhanced migration of cells through modulation of CXCR4; however, these cells may adopt a cancer-like phenotype following hypoxic preconditioning due to accumulation of reactive oxygen species . Amongst all strategies, surface modulation of CXCR4 is widely accepted to help in tissue-directed migration of MSCs mediated by SDF-1α [39, 52–54]. SDF-1α is significantly upregulated in almost all injured tissues, which facilitates the migration and engraftment of circulating CXCR4-positive cells . Therefore, it is believed that inadequate amounts of CXCR4 on the MSC surface may be responsible for the cells’ insufficient migration and homing towards the injured site.
We reported previously that IL-3 inhibits differentiation of human hematopoietic stem cells into bone-resorbing osteoclasts, and also enhances the differentiation of human BM-MSCs into bone-forming osteoblasts under both in-vitro and in-vivo conditions [30, 56]. These results indicated that IL-3 enhances the regenerative potential of human MSCs and is a promising therapeutic candidate for repair or prevention of tissue damage. In the present study, we investigated the role of IL-3 on migration of human MSCs under both in-vitro and in-vivo conditions. Although MSCs are found in many tissues, studies comparing the migration potential of MSCs isolated from different sources are limited. Therefore, we first examined the expression of IL-3Rα on BM-MSCs, AT-MSCs and GT-MSCs and found that all three sources of MSCs express IL-3Rα at gene and protein levels.
Wound healing assay revealed that exposure of MSCs to IL-3 significantly enhances cell migration and wound closure. Cell motility is an important parameter to study the cell migration. Time-lapse microscopic studies of human MSCs in the presence of IL-3 showed significant increase in their motility as evidenced by increased euclidean and accumulated distances. Transendothelial cell migration is a multistep process initiated by firm adherence of cells to the endothelium via selectins, followed by multiple cascades of chemokine and integrin signaling. Human MSCs are known to express a set of chemokine receptors and integrins that are functionally required for cell migration . Engagement of CD44 with its ligand hyaluronic acid or E-selectin induces the firm adhesion and subsequent transendothelial migration . Analysis of chemokine profile of human MSCs showed that IL-3 does not affect the expression of chemokine receptors such as CCR1, CCR7, CCR9, CX3CR1, CXCR5, CXCR6, integrins α4 and α5 and CD44 on MSCs. Interestingly, the expression of CXCR4 was significantly and consistently increased by IL-3 in all three MSCs at both surface as well as intracellular levels. Increased intracellular expression of CXCR4 might serve as a receptor reservoir , which can be displayed to cell surface in response to IL-3. Further, real-time PCR analysis revealed 6-fold to 8-fold upregulation of CXCR4 in human MSCs upon IL-3 treatment.
To further confirm the effect of IL-3 on CXCR4 expression, we examined the migration of IL-3-treated MSCs towards SDF-1α. A greater number of IL-3-treated MSCs migrated towards SDF-1α, suggesting its pivotal role in enhancing CXCR4 expression. SDF-1α-mediated migration is inhibited by CXCR4 antagonist AMD3100, indicating the key role of the CXCR4/SDF-1α axis in MSC migration . IL-3-induced CXCR4-mediated migration of MSCs towards SDF-1α was decreased in the presence of AMD3100. Similarly, AMD3100 decreases the migration of IL-3-treated MSCs towards the wound area. These results suggest involvement of the CXCR4/SDF-1α axis in IL-3-induced MSC migration. The therapeutic impact and feasibility of the IL-3 preconditioning approach was evaluated in vivo using a matrigel-releasing SDF-1α implantation assay in NOD/SCID mice. The combination of SDF-1α with matrigel renders the current approach to be of therapeutic relevance, because it generates a SDF-1α gradient, mimicking tissue injury in vivo. Interestingly, IL-3 pretreatment enhances in-vivo migration of MSCs towards SDF-1α.
Comparative study for induction of CXCR4 expression by different cytokines revealed that IL-3 is equally as potent a cytokine as TNF-α and is more potent than IL-1β. Unlike IFN-γ, IL-3 does not alter the phenotypic and immunophenotypic characteristics of MSCs. Additionally, in contrast to TNF-α and IL-1β, IL-3 possesses anti-inflammatory and immunomodulatory properties even in the presence of severe inflammatory conditions and prevents tissue damage in animal models [28, 29, 60]. All of these results suggest that compared to other cytokines IL-3 has the added advantage of its anti-inflammatory and immunomodulatory properties, and it increases both the migration and regenerative potential of human MSCs under both in-vitro and in-vivo conditions. Further we observed that IL-3 does not affect the proliferation of human MSCs and it is not toxic to cells even at higher concentrations (Additional file 1: Figure S2). To further confirm that IL-3 is not toxic in vivo, we evaluated the toxicity of IL-3-treated human MSCs in SCID mice. We found that all hematological parameters and the differential blood cell count were unchanged by infusion of IL-3-treated MSCs (Additional file 1: Table S1). Also, IL-3-treated MSCs did not show any adverse effect on various vital organs (data not shown). In addition, we found that IL-3-treated human MSCs were nontumorogenic in mice (Additional file 1: Figure S3). Thus, IL-3 preconditioning seems to be a promising strategy for improvement of site-directed migration of MSCs and their tissue regeneration potential in vivo. Overall, IL-3 has more therapeutic potential than other cytokines in regenerative cell therapies. Thus, we demonstrate for the first time that pretreatment of MSCs with IL-3 is a novel strategy to achieve better therapeutic outcomes for the cells in tissue regeneration.