Differential expression of transcription factors E2Fs in human pluripotent stem cells and in its differentiated progeny into neural lineage

MARÍA SOLEDAD RODRÍGUEZ VARELA; VERONICA FRUMENTO; GUILLERMO VIDELA RICHARDSON; NICOLAS ALEXIS DIMOPUOLOS; SANTIAGO G. MIRIUKA; GUSTAVO SEVLEVER; MARÍA ELIDA SCASSA; LEONARDO ROMORINI

Mar del Plata

Congreso; SAIC SAI SAFE 2016; 2016

SAIC

The embryonic or induced human pluripotent stem cells (hPSCs) have an unusual cell cycle structure which consist of a short G1 phase and absence of G1/S checkpoint. The E2F transcription factors have been shown to be critical for the temporal expression of the oscillating cell cycle genes. In somatic cells activators (E2F1, E2F2 and E2F3) and represors (E2F3B, E2F4 and E2F5) E2Fs show a periodic expression profile. The activators E2Fs have the higher levels of expression in the S phase while the major expression of represors E2Fs is in the G2/M phase. In hESCs, the role of the various complexes RB/E2F and of the factors that determine which binds each complex still remains uncertain. There are few reports on how the expression of these transcription factors is in pluripotent cells. In this study we determinated by flow cytometry that PSCs have an S phase cell population of 43,5±12%, whereas their differentiated progeny into neural lineage (Nestin+, Neurofilament light chain+, Doublecortin+) have one of 9,2±0,7% similar to that present in fibroblasts (10%). At the same time, we studied the expression levels of certain E2Fs throughout the cell cycle in PSCs and neural progenitors (NP). By RT-qPCR we determinated that PSCs express higher levels of the E2F1, E2F3, E2F4 and E2F5 transcripts in contrasts somatic cells (fibroblasts). The expression of ARNm of E2F2 and E2F3B in hESCs did not differ with somatic cells whereas iPSCs show high levels of expression of both transcripts. NP have a similar profile to hESCs from wich they derive, except E2F2 mRNA, being its expression higher in the NP. To determine if the analyzed E2Fs are expressed periodically we synchronize the cells in G1/S with PD0332991, specific inhibitor of CDK4/6 (30h 5µM for PSC and 24h 1µM for NP) and in G2/M with nocodazole (24h 100ng/ml for PSC and 54h 200ng/ml for NP). RT-qPCR analysis revelead significant differences in the expression levels of the E2Fs transcripts suggesting a periodic expression profile.