Effects of Glucocorticoid Receptor Small Interfering RNA Delivered Using Poly Lactic-Co-Glycolic Acid Microparticles on Proliferation and Differentiation Capabilities of Human Mesenchymal Stromal Cells

Abstract

Bone marrow-derived mesenchymal stem cells (MSC) are a potential attractive source of cells for stem cellbased tissue regeneration, but the small number and reduced capabilities of MSC proliferation and differentiation due to in vitro replicative senescence and donor-associated pathophysiological factors, including age and estrogen depletion, severely restrict their potential usefulness in clinical applications. Glucocorticoids (GC) are well-known steroid hormones that regulate MSC proliferation and differentiation, but the defined effects and underlying mechanisms of endogenous glucocorticoids on MSC characteristics are not understood. This study investigated the effects of the blockage of endogenous GC using glucocorticoid receptor (GR) small interfering RNA (siRNA) delivered using biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles on proliferation and differentiation capabilities of human MSC in vitro. The results show that we can prepare PLGA microparticles as a delivery system for GR siRNA and maintain release of siRNA up to 40 days in vitro. Transfection of GR siRNA significantly downregulates GR and upregulates the expression of fibroblast growth factor-2 and Sox-11 of human MSC. MSC that have proliferated with endogenous GC blocked in vitro have greater proliferation rates and exhibit upregulated expression of osteogenic markers (alkaline phosphatase and core binding factor alpha 1) under differentiation stimulation after 1 week. Under adipogenic differentiation, MSC proliferated in vitro with siRNA transfection, resulting in significantly lower adipogenic markers (peroxisome proliferatoractivated receptor and lipoprotein lipase) than controls. In conclusion, PLGA particles can serve as a tool for delivery of GR siRNA to effectively block the effects of endogenous GC on MSC, which has the potential to improve the capabilities of human MSC for clinical application by preventing replicative senescence.

Glucocorticoid receptor antagonist and siRNA prevent senescence of human bone marrow mesenchymal stromal cells in vitro

“…1a). Our previous studies have demonstrated that the treatment with GR siRNA-loaded PLGA particles knocked down the expression of GR mRNA in MSCs and the GR mRNA level returned to near control levels after 5 to 7 days [26] . No significant difference of GR mRNA expression was observed in the MSCs treated with RU486 or GR siRNA compared to controls after 4 weeks when MSCs started to differentiate (Fig.…”

“…The GC responses in MSCs are mediated via type 2 intracellular GR, and hence blockage of the effects mediated by GCs contained in culture medium using GR siRNA or RU486 has been shown to improve the capability for MSC proliferation and increase the expression of osteogenic differentiation markers [25–26] . The present study further demonstrates that both of these distinct treatments up-regulate the expression of telomerase mRNA, increase antioxidant levels, and increase MSC telomere length during in vitro expansion.…”

“…MSC DNA content was measured using a DNA quantification kit (FluoReporter ® Blue Fluorometric dsDNA Quantitation Kit, Invitrogen) according to the manufacturer’s protocols. The difference in DNA content after every 48hrs was used to determine the MSC proliferation rate as described in our previous studies [25–26] . NR3C1 level after different treatments were measured using a commercially available ELISA kit (MyBioSource, San Diego, CA).…”

“…Elevated levels of endogenous GCs also contribute to MSC senescence and the development of osteoporosis in aging patients and in those who are postmenopausal and hence estrogen depleted [12–24] . Our previously published data have shown that short-term blockage of the effects mediated by the GCs present in fetal bovine serum (FBS) using either the GR antagonist RU486, or the small interfering RNA (siRNA) for GR, significantly improve the proliferative and osteochondral differentiation capabilities of human MSCs [25–26] . However, because of the short-term nature of these previously conducted experiments, we were unable to assess the potential effectiveness of both of these treatments on the senescence and antioxidant activity of these stem cells.…”

Small non-coding RNAs-based bone regulation and targeting therapeutic strategies

“…We have discussed the role of glucocorticoids (GCs) in osteoblastogenesis and osteoclastogenesis in this review; therefore, designing siRNA targeting GC receptor (GCR) could be a feasible approach for regulating the function of hBMSCs, which was explored in an in vitro study (Wei et al, 2013). Moreover, Hong et al reported that GCR-siRNAdelivered PLGA microparticles showed effectively inhibited, detrimental effects of endogenous GC on hBMSCs, with improved osteogenic proliferation and differentiation (Hong et al, 2012). Wang et al fabricated a site-specific bone-targeting system based on an octa-aspartic acid sequence (Asp8) and bisphosphonates, which was demonstrated to favorable recognize and strongly bind to biomineral (e.g.…”