Abstract
BackgroundThe pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved.In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming).MethodsImmuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol.ResultsThe kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes.ConclusionThis study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.
Highlights
The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo
For the genomic cDNA sequencing, analysis of statistical differences in gene levels in cells from the 24- and 48-h primed and non-primed MPC cultures were determined using the manufacturers’ software with q values < 0.045 being accepted as Kinetics of binding and uptake of PPS by MPCs in culture The kinetics of binding and uptake of PPS by cultured MPCs when added to the media at concentrations of 0.5–10 μg/ml was monitored by the percentage decrease in their media levels over 24 h using the PPS enzyme-linked immunosorbent assay (ELISA)
These observations suggest a rapid binding of PPS to cell surface heparin receptors, followed by a time- and concentration-dependent shedding and uptake by the MPCs over the 24 h of culture [28, 29]
Summary
The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. Adult mesenchymal stem cells (MSCs) are an abundant source of self-renewing, multipotent undifferentiated cells that can be readily isolated from bone marrow, adipose tissue, muscle, and synovium. They can be serially expanded in culture and cryopreserved almost indefinitely without significant loss of their tissue regenerative capacity [1,2,3,4]. When administered systemically, MSCs exhibit the capacity to migrate to the site(s) of tissue injury, where they can modulate inflammatory and immune-regulatory pathways as well as release pro-anabolic factors [6,7,8,9] These unique activities of MSCs have led to extensive investigations into their potential applications as biological agents for the treatment of a variety of clinical applications [5,6,7]. MSCs have been considered a suitable therapy for muscular skeletal and connective tissue disorders, including degenerative disc disease, osteoarthritis, and repair of articular cartilage, owing to the high incidence of such disorders as well as their limited capacity for spontaneous repair and the limited treatment options [10,11,12,13,14,15,16]
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