Differential Cyclin Dependent Kinase Inhibitors Expression in Human Embryonic Stem Cells

VIDELA RICHARDSON G; SCASSA M; QUESTA M; FERNANDEZ ESPINOSA D; RISSO M; LOSINO N; LUZZANI C; HEYD V; GUBERMAN A; SEVLEVER G; MIRIUKA S

Barcelona, España

Congreso; ISSCR 7th Annual meeting; 2009

ISSCR

CDK inhibitors (CKIs) regulate early stages of cell cycle progressionby inhibiting G1/S CDK-cyclin complexes. In mammals CKIs are dividedinto two families: INK4 which includes p16INK4a, p15INK4b, p18INK4c and p19INK4dand Cip/Kip , which comprises p21Cip1, p27Kip1 and p57Kip2. A large body ofevidence suggests that the cellular decision to proliferate, differentiate,apoptose, become quiescent or enter into senescent arrest is often made inthe G1 phase. Human embryonic stem cells (hESC) have an unusual divisioncycle with a short G1 phase. Oxygen is a critical component of the embryonicenvironment and changes in oxygen availability can influence both embryonicgene expression and subsequent fetal development. The aim of this studywas to analyze the expression of CKIs in stemness, during differentiationand under hypoxic conditions. Methods: hESC (H5 and H9 lines) were grownon irradiated MEF (iMEF) feeder layer in KO-DMEM medium supplementedwith 10% KSR and 4 ng/ml bFGF. Alternatively, cell were grown on Matrigelwith iMEF conditioned medium. Pluripotency was confirmed by the expressionof stemness markers. Differentiation was induced by the embryoid bodyformation method in DMEM supplemented with 20% fetal bovine serum.Hypoxic conditions were generated in a sealed chamber insufflated with a gasmixture containing 95% N2 and 5% CO2. Results: By RT-PCR and westernblot analysis we determined the expression of CKIs during stemness and afterinducing differentiation in H5 and H9 hESC lines. We found that p18INK4c,p19INK4d, p21Cip1, and p27Kip1 were minimally expresse d in undifferentiatedhES, but induced upon the onset of differentiation. Interestingly, p16INK4a andp15INK4b were present in both cell lines in the undifferentiated state and duringdifferentiation. Notably, p57Kip2, although highly expressed in undifferentiatedas well as in differentiated cells, was significantly more abundant in theH5 cell line. CKIs expression analysis under hypoxia revealed a significantincrease in p21Cip1 suggesting a role for CKIs at cellular adaptation to hypoxia.Conclusions: We determined that most CKIs are expressed in very low levelsin the undifferentiated state, but induced upon differentiation. However,p15INK4b, p16INK4a and p57Kip2 are significantly expressed in undifferentiatedcells. Nevertheless, p57Kip2 is much more abundant in undifferentiated H5 cellsthan in H9 counterparts, revealing the existence of differences in the expressionof cell cycle regulatory components between lines. Additionally, in hESCp21Cip1 expression is also up-regulated by hypoxia. Considering the uniqueproliferative properties of hESC, the presence of p15INK4b, p16INK4a and p57Kip2 isintriguing. Given the emerging individual roles of CKIs in fundamental cellularprocesses, we hypothesize that the presence of certain CKIs during stemnessmay be required for as yet unidentified functions, probably unrelated with cellcycle halt.