Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Abstract

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate

“…In untreated hESC, the expression of SSEA-4 is widely acknowledged (most cells express SSEA-4) [33, 34], but as we have shown previously, expression of SSEA-3 correlates more precisely with coexpression of NANOG and OCT4 [33]. Furthermore, by utilising SOX2 detection, two subpopulations of hESC could be characterized: SSEA-3+NANOG+OCT4+SOX2+ cells and SSEA-3+NANOG−OCT4−SOX2+ cells.…”

Increased Culture Density Is Linked to Decelerated Proliferation, Prolonged G1 Phase, and Enhanced Propensity for Differentiation of Self-Renewing Human Pluripotent Stem Cells

“…Recent findings [25] corroborate ours on the lengthening of the G 1 phase suppressing hPSC selfrenewal and leading to more extensive differentiation. Hence, controlling, in part, the propensity for commitment through modulation of the cell cycle may be an appealing alternative to growth-arresting agents, such as nocodazole and DMSO, which often induce aberrant differentiation and apoptosis [8,36]. Cells at a low or high density display distinct profiles of the CDK inhibitor p21 (CDKN1A), warranting further investigation into its participation in molecular mechanisms regulating self-renewal or commitment.…”

“…However, the cycle variability of hPSCs in conditions supporting their self-renewal has received less attention. Interestingly, the fractions of hPSCs in the G 1 phase reported in different studies are not consistent [1,3,[7][8][9]. Considering that cell cycle duration is intimately linked to proliferation kinetics, the wide range of doubling times (T d ; 18 to over 60 h) of hPSCs in many studies [10][11][12] also exemplifies the variability of proliferation and cycle of self-renewing hPSCs.…”

Human Embryonic Stem Cells Exhibit Increased Propensity to Differentiate During the G1 Phase Prior to Phosphorylation of Retinoblastoma Protein

“…It may also be caused by the arrest itself irrespective of phase or even by direct effect of CDK2 on pluripotency of hESCs [10]. Similar concerns apply to studies which showed that chemical arrest of the cell cycle in G2 using Nocodazole also promotes the differentiation of hESCs [18]. Thus, a causal relation between the G1 phase and differentiation of hESCs has not yet been fully established, and it remains unclear whether hESCs are more sensitive to differentiate in their G1 phase versus other phases.…”