189 SMALL MOLECULE INHIBITORS PD0325901 AND CHIR99021 CAUSE REDUCED EXPRESSION OF PLURIPOTENCY GENES IN PUTATIVE PORCINE INDUCED PLURIPOTENT STEM CELLS

Abstract

Small molecule inhibitors acting on the MEK and Wnt signalling pathways (PD0325901 and CHIR99021, respectively) have been used successfully for the maintenance of murine and rat pluripotent stem cells in the in vitro culture. The effects of these compounds in other species have not been conclusively determined, and some reports suggest that they may actually cause loss of pluripotency in human embryonic stem cells and decrease of OCT4 expression levels in porcine induced pluripotent stem cells (piPSC). In our experiments, putative piPSC lines derived from transgenic porcine fetal fibroblasts (pFF) and adipose mesenchymal stem cells (pAMSC) that harbor the OCT4-EGFP reporter construct were maintained in piPSC culture medium [DMEM supplemented with 10% Knockout Serum Replacement (Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin-streptomycin, and 1000 U mL–1 murine leukemia inhibitory factor (LIF)], with or without addition of 1 μM PD0325901 and 3 μM CHIR99021. After five passages in the two experimental culture conditions, three pFF-derived putative piPSC lines were evaluated morphologically and the expression of various pluripotency genes was analysed by real-time quantitative PCR. The results showed decrease of OCT4-EGFP expression and loss of compact colony morphology of the cells cultured in the presence of PD0325901 and CHIR99021. Moreover, the transcriptional expression levels of OCT4, SOX2, NANOG, REX1, UTF1, and TDH were reduced by 12-, 9-, 10-, 3-, 5-, and 20-fold, respectively, compared with the cells cultured without these inhibitors. When putative piPSC derived from pAMSC were cultured in medium supplemented with small molecule inhibitors, the OCT4-EGFP expression was completely lost within a few days. The cells also lost their iPSC-like colony morphology and were further propagated as single, mesenchymal-like cells. The same effects were observed when the cells were cultured with CHIR99021 alone, whereas there were no such changes when we used only PD0325901. This suggests that, similarly to human embryonic stem cells (ESC), the activation of the Wnt pathway in pAMSC-derived iPSC may lead to differentiation in these cells. The effects of the CHIR99021 alone on the pFF-derived piPSC-like cells are still to be determined. In conclusion, the results of our preliminary investigations call into question the effectiveness of PD0325901 and CHIR99021 in the maintenance of piPSC in culture.