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

VIDELA RICHARDSON G; SCASSA ME; QUESTA M; FERNANDEZ ESPINOSA D; RISSO M; LOSINO N; LUZZANI C; HEYD V; GUBERMAN AS; SEVLEVER GE; MIRIUKA SG

Barcelona, España

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

International Society of Stem Cell Research

Eukaryotic cell cycle progression is dependent in part, on the tightly regulated activity of cyclin-dependent kinases (CDKs). CDK4/6 and CDK2 whose regulatory partners are the D-type cyclins and cyclin E respectively, represent two different classes of G1-specific CDKs, whose activation is required for entry into S phase. Two class of molecules, defined based on their evolutionary origins, structure and CDK specificity, can bind to CDKs and inhibit their kinase activity .One class is the INK4 family, which includes p16INK4a, p15INK4b, p18INK4c and p19INK4d. The second group is the Cip/Kip family composed of p21Cip1, p 27Kip1 and p57Kip2. A large body of evidences suggest that the cells decision to differentiate, proliferate, apoptose, become quiescent or enter into senescent arrest is often made in the G1 phase Embryonic stem cells (ESCs) are pluripotent, they can replicate indefinitely and differentiate into multiple tissues from all three embryonic germ layers. Due to their unique properties, ESCs serve as a model system for studying embryo development and hold great promise for regenerative medicine. ESCs have a peculiar cell cycle. One key difference between somatic and ESCs is the length of G1 phase. Their cell division rates are very short and exhibit a short G1 phase. To date, little is known about cell cycle regulation in ESCs and how the cell cycle machinery influences their properties and their fate commitment. Firstly, we sought to determine the expression pattern of CKIs under experimental conditions that maintain pluripotency or induce differentiation in H5 and H9 human ESCs lines. By RT-PCR and western blot analysis we found that p18INK4c, p19INK4d, p21Cip1, and p 27Kip1were almost undetectable in undifferentiated hESC and were induced during the course of their differentiation. Interestingly p16INK4aand p15INK4b were present in both cell lines, in pluripotent cells and in embryoid bodies (EBs) along differentiation. Importantly p57Kip2 seemed to be more abundant in H5 ESC line. Notably, H5 cells display an increased propensity to develop contractile EBs under differentiation conditions. The mentioned EBs express cardiac markers such as GATA-4, Nkx2.5, Isl-1, cTnT and ANP and respond to norepinephrine. Oxygen is a significant component of the embryonic environment and reductions in oxygen availability can influence both embryonic gene expression and subsequent fetal development .Taking into account the aforementioned considerations, we also explored the expression of CKIs under hypoxic conditions. We found that p21Cip1 was induce in pluripoten cells exposed to hypoxia poner resultadosy conclusion The present study reveals some differences between human ESC lines in the expression of cell cycle regulatory components which has not been described earlier. Our data suggest some intrinsic link between CKIs and cell fate decision among hESC lines (differentiation into a specific lineage) It is our challenge to elucidate the role of individual CKIs in the maintenance of ESCs pluripotency and the processes that govern ESCs fate decisions. ..