Supplementary MaterialsSupplemental Material kccy-17-14-1496740-s001. involves the activation Dp44mT of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a Rabbit Polyclonal to ACRBP mechanism that requires a functional CDK2 but not GSK3 activity. Abbreviations: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III -tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium models for human development studies, disease modeling and drug discovery [1,2]. The ability of hPSCs to maintain their self-renewal and pluripotency is usually associated with their capacity to remain in a proliferative condition [3,4]. To achieve this, hPSCs are endowed with an atypical cell cycle which lacks fully formed G1 and G2 gap phases and in which a high proportion of time (60%) is usually devoted to DNA replication (S phase). While there are exceptions, hPSCs generally have short generation occasions of 8C16?hours [5C7]. Importantly, when hESCs initiate a differentiation process, cells accumulate in the G1 phase and lengthen their cell cycle (more than 16?hours) [8]. A short G1 phase limits the time in which hPSCs can be influenced by external differentiation signals, as this phase represents the time with increased sensitivity to differentiation cues [9,10]. Moreover, it has been exhibited that inhibiting progression of the G1 phase commits hESCs differentiation [11,12]. The transition from one cell cycle phase to another is usually governed by key regulators called cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases which are activated at specific points during cell cycle and whose actions are dependent on their associations with regulatory subunits, named cyclins [13,14]. The levels of cyclins in different stages of the Dp44mT cell cycle differ, whereas the CDK protein levels remain stable [14]. In particular, somatic cells cycle progression from G1 to S phases depends in part of the fine regulation of the activity of the complexes formed by the catalytic subunits CDK2, CDK4 and CDK6, whose expression levels remain constant, and the regulatory subunits, D-type Dp44mT (D1, D2 and D3) and E cyclins, whose expression levels oscillate during the cell cycle [15]. The activities of the cyclin/CDK complexes are counteracted by the action of small polypeptides, the CDK inhibitors (CKIs) [16,17]. However, in murine embryonic stem cells (mESCs) most cyclins and CDKs are expressed throughout the cell cycle. High levels of CDKs activities in mESCs are due in part to the absence or low expression of CKIs, which in turn are linked to the high levels of Dp44mT cyclins that remain present during the cell cycle [18]. At present, to the best of our knowledge there is only a limited amount Dp44mT of information referred to cell cycle regulation in hPSCs and most of it relies on hESCs. Although, under certain circumstances, these cells exhibit a cell cycle similar to mESCs [18], differences in cell cycle control between hESCs and mESCs are evident, and have motivated several groups to study the expression profiles of key cell cycle regulators in hESCs [19]. In particular, and in contrast to mESCs, most cell cycle regulators in hESCs exhibit a phase-specific expression [20]. However, there are discrepancies between results from different research groups regarding the abundance and periodic or constitutive expression of some cyclins during hESCs cell cycle [3,5,9,20C23]. Besides, there is not.