Figure 8.
Effect of mesenchymal stem cell (MSC) pretreatment with polyinosine-polycytidylic acid [Poly (I:C)], a toll-like receptor 3 (TLR3) agonist, on messenger RNA (mRNA) expression for cyclooxygenase 2 (COX2) and IL-10 in MSCs and its released microvesicles (MVs) and in monocytes exposed to MVs released by Poly (I:C)–pretreated MSCs (MSC MVs). In these experiments, total RNA was extracted from human MSCs (B, C), MSC MVs (D), or human monocytes (E, F), and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed. Top: Representative agarose gels of semiquantitative RT-PCR products from COX2 (B, D, E) and IL-10 (C, F) mRNA amplification. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control to normalize loading of the RNA samples. The PCR products were 295 bp in size for COX2 and 500 bp for IL-10. Bottom: Densitometry readings derived from the PCR gels. The band density relative to that of the GAPDH is expressed as mean ± SD. (A) Human MSCs expressed TLR3. (B, C) Human MSCs were cultured with or without Poly (I:C) for 1 hour and serum starved for 48 hours. TLR3 stimulation further increased the expression of COX2 and IL-10 mRNA in MSCs [n = 8–22 for standard control (STD MSCs) and Poly (I:C)–stimulated MSCs]. *P < 0.01 by Student’s t test. (D) TLR3 stimulation also further increased the mRNA expression for COX2 in the released MVs, Poly (I:C) MVs (n = 10 for MVs released from standard MSCs [STD MVs] and MVs released from MSCs prestimulated with Poly (I:C) MVs [Poly (I:C) MVs]). *P < 0.01 by Mann–Whitney test. (E, F) mRNA expression for COX2 and IL-10 was increased in human monocytes exposed to Poly (I:C) MVs (n = 5–8). *P < 0.01 by Student’s t test.