The In Vitro Migration Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells in Response to Chemokines and Mesenchymal Growth Factors.

Abstract

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.