Effect of Aspirin and Salicylic Acid on LPA Induced Differentiation of P19 Stem Cells into Cardiomyocytes

Abstract

The use of stem cell-based therapy in conjunction with existing medical interventions, to target complications caused by coronary artery disease (CAD) is not fully examined. In parallel, the role of lysophosphatidic acid (LPA); an important endogenous bioactive phospholipid, has shown cardioprotective characteristics at low physiological concentrations, providing a potential for future treatment plans. In addition, studies have indicated the promise of aspirin (ASA)/ salicylic acid (SA) or LPA to induce and promote cardiac differentiation of SCs in various models. Therefore, in this project, we investigated the effects of ASA/SA in the presence or absence of LPA to induce the differentiation of the murine P19 teratocarcinoma stem cell line into cardiomyocytes. Routine cell culture was undertaken using P19 stem cells cultured in complete α-minimal essential medium (α-MEM). In the first instance, the protocol was optimised to ensure that efficient and reproducible differentiation was achieved. Embryoid bodies (EB) were formed by seeding cells and left to aggregate over a period of 2 days in ultra-low attachment 96-well plates, to establish differentiation. P19 stem cells were pre-incubated for 1 hour with ASA and SA at varying concentrations (0.1mM, 0.3mM, 1mM and 3mM) and selective NFκB inhibitor (0.1nM CAY10470) were pre-incubated 1 hour prior to adding LPA (5µM). Control cells were cultured in complete α-MEM alone. 6-8 EBs were isolated and seeded into 12-well tissue culture plates and cultured for 6 days. Western blotting was used to confirm differentiation, examining for the expression of ventricular myosin light chain (MLC-1v), relative to β-actin. To determine the potential mechanism through which differentiation may be induced, changes in phosphorylation of activated NFκB and IκB were determined. Optimisation of the differentiation protocol revealed that 1 x 104 cells grown for 2 days, produced consistent EBs sizes which ranged between 350-450µm in diameter. These EBs efficiently differentiated into cardiomyocytes. Differentiation was consistently achieved using LPA (5µM) and at selected concentrations of ASA (0.3 -1mM, at day 3) and SA (1mM, at day 3). Maximal expression of MLC-1v in ASA/SA conditions was seen at 1mM. However, LPA induced differentiation was inhibited by both in combination treatment with ASA and SA, despite both inducing differentiation independently. Analysis of phosphorylated and native proteins associated with the NFκB complex was successfully detected. These initial studies indicated substantial expression of phospho NFκB in LPA, SA and ASA treated cells and increases were seen at the 6-9-hour time points. The expression of phospho IκB in LPA treated cells peaked at 10-15 mins, while ASA/SA treated cells showed phospho IκB peaking at a later time point (3 hours). In conclusion, the experiments conducted in this thesis have shown that both ASA/SA and LPA induced cardiomyocyte differentiation. However, when ASA or SA are used in combination with LPA, an antagonistic effect is seen, preventing LPA to induce differentiation.