Enhanced mir-145 Expression in Cardiomyocytes Generated From Lysophosphatidic acid (LPA) Induced P19 Stem Cells

Lysophosphatidic acid (LPA) is an endogenous bioactive molecule with the potential to regulate stem cell differentiation into multiple lineages. The underlying mechanisms through which this may be mediated are however currently poorly understood. In particular, it is not clear whether LPA regulate micro RNA (miRNAs) expression or change their profile during lineage commitment or after differentiation. Thus, studies have been initiated to confirm whether LPA induces stem cells to differentiate into cardiomyocytes and further establish whether this may be associated with changes in miRNA expression, focussing initially on mir-145 since the latter is very low in embryonic stem cells but increases in the differentiated cells and has been reported to suppress pluripotent transcription factors.

All studies were carried out using the P19 mouse carcinoma stem cell line maintained in α-MEM supplemented with 10% foetal bovine serum plus 1% penicillin and streptomycin. To induce differentiation, cells were plated together with LPA (0.5, 1, 5, 10 and 20 µM or 5 µM alone) in non-adherent petri dishes over a period of 4 days to form embryoid bodies (EBs). Controls were incubated with culture medium alone in the absence of LPA. Subsequently, EBs were transferred into adherent tissue culture grade 6-well plates and monolayers were lysed at 0, 3, 6, 9, and 12 days after plating. Differentiation of P19 stem cells to cardiomyocytes was confirmed by visualisation of beating clusters under an inverted Olympus (CKX41) microscope. Cell lysates were subjected to western blotting for cardiac ventricular myosin light chain (MLC-1v) expression, troponin I or OCT4 expression. To determine changes in mir-145 expression, qRT-PCR analysis of total RNA isolated from controls and LPA-treated cells was carried out using the mirVana™ kit following manufacturers protocol. Two control house-keeping genes U6 and β-actin were selected for normalisation.

Incubation of P19 cells with LPA induced their differentiation into cardiomyocytes. This was confirmed by the induction of MLC-1v and toponin I expression which increased with increasing LPA concentration with 5 µM producing the optimum effect (p<0.01). These changes were accompanied by beating clusters of differentiated cells. The changes observed were also time dependent with MLC-1v expression reaching a peak at day 6. In other studies carried out, expression of OCT4 decreased in parallel with the increase in expression of the cardiac specific markers and this was significant with 1, 5, 10 and 20 µM LPA. In parallel experiments 5 µM LPA upregulated mir-145 by 2 fold above controls in cardiomyocytes generated from the P19 cells.

The data obtained from our studies thus far confirm that at physiological levels (2-20 µM) LPA is able to induce the differentiation of P19 stem cells into cardiomyocytes in a concentration and time dependent manner. Additionally, mir-145 is upregulated by LPA in induced cardiomyocytes suggesting that it may be critical in mediating the effects observed with LPA. Further studies are however required to confirm this and establish whether induction of mir-145 expression precedes lineage commitment or mediate differentiated cardiomyocyte survival and proliferation. This and other experiments looking at changes in additional miRNAs are currently being conducted including work on specific LPA receptors antagonist and miRNA inhibitors to analyse how effective are those changes to the cells.