Differential cyclin dependent kinase inhibitors expression in human embryonic stem cell lines

GUILLERMO VIDELA RICHARDSON; MARÍA ELIDA SCASSA; MARÍA QUESTA; DARÍO FERNÁNDEZ ESPINOSA; MARIQUENA RISSO; NOELIA LOSINO; CARLOS LUZZANI; VANESA HEYD; ALEJANDRA S. GUBERMAN; GUSTAVO SEVLEVER; SANTIAGO G. MIRIUKA

Barcelona, España

Congreso; International Society of Stem Cell Research 7th Annual Meeting; 2009

International Society of Stem Cell Research

Background: CDK inhibitors (CKIs) regulate early stages of cell cycle progression by inhibiting G1/S CDK-cyclin complexes. In mammals CKIs are divided into two families: INK4 which includes p16INK4a, p15INK4b, p18INK4c and p19INK4d and Cip/Kip which comprises p21Cip1, p27Kip1 and p57Kip2. A large body of evidence suggests that the cellular decision to proliferate, differentiate, apoptose, become quiescent or enter into senescent arrest is often made in the G1 phase. Human embryonic stem cells (hESC) have an unusual division cycle with a short G1 phase. Oxygen is a critical component of the embryonic environment and changes in oxygen availability can influence both embryonic gene expression and subsequent fetal development. The aim of this study was to analyze the expression of CKIs in stemness, during differentiation and under hypoxic conditions. Methods: hESC (H5 and H9 lines) were grown on irradiated MEF feeder layer in KO-DMEM medium supplemented with KSR 10% and bFGF 4 ng/ml. Alternatively, cell were grown on Matrigel with iMEF conditioned medium. Pluripotency was confirmed by the expression of stemness markers. Differentiation was induced by the embryoid body formation method in DMEM supplemented with fetal bovine serum 20%. Hypoxic conditions were generated in a sealed chamber insufflated with a gas mixture containing 95% N2 and 5% CO2. Results: By RT-PCR and western blot analysis we determined the expression of CKIs during stemness and after inducing differentiation in H5 and H9 hESC lines. We found that p18INK4c, p19INK4d, p21Cip1, and p27Kip1 were minimally expressed in undifferentiated hES, but induced upon the onset of differentiation. Interestingly, p16INK4aand p15INK4b were present in both cell lines in the undifferentiated state and during differentiation. Notably, p57Kip2, although highly expressed in undifferentiated as well as in differentiated cells, was significantly more abundant in the H5 cell line. CKIs expression analysis under hypoxia revealed a significant increase in p21Cip1 suggesting a role for CKIs in cellular adaptation to hypoxia. Conclusions: We determined that most CKIs are expressed in very low levels in the undifferentiated state, but induced upon differentiation. However, p15INK4b, p16INK4a and p57Kip2 are significantly expressed in undifferentiated cells. Nevertheless, p57Kip2 is much more abundant in undifferentiated H5 cells than in H9 counterparts, revealing the existence of differences in the expression of cell cycle regulatory components between lines. Additionally, in hESC p21Cip1 expression is also up-regulated by hypoxia. Considering the unique proliferative properties of hESC, the presence of p15INK4b, p16INK4a and p57Kip2 is intriguing. Given the emerging individual roles of CKIs in fundamental cellular processes, we hypothesize that the presence of certain CKIs during stemness may be required for as yet unidentified functions, probably unrelated with cell cycle halt.