Self‐renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Abstract

Competency for self-renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH-designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15-16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT-2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220(NPAT) containing Cajal bodies revealed that the durations of the S ( approximately 8 h), G2 ( approximately 4 h), and M phases ( approximately 1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti-tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5-3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT-PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin-dependent kinases (CDKs), we observed high mRNA levels for the G1-related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication.

Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells

“…In contrast to mouse embryonic stem cells, which are characterized by a higher CDK6 expression and lack of CDK4 activity (Faast et al, 2004), our investigation of hESCs demonstrated that expression of CDK4 is higher than that of CDK6, in agreement with an earlier publication (Becker et al, 2006). In addition, CDK2 kinase activity is higher than that of CDK4 and CDK6, which may reflect the formation and a turnover of several complexes such as CDK2-CYCLIN E, CDK2-CYCLIN A, CDK2-CDC25A and CDK2-c-MYC as revealed by our immunoprecipitation studies.…”

“…On day 3 after plating, hESCs cultured on Matrigel-coated plates were synchronized at the G2 stage of the cell cycle by incubation for 16 h with 200 ng/ml nocodazole (Sigma-Aldrich Ltd, Dorset, UK) as described by Becker et al (2006). For collection of cells at the G1 stage of the cycle, cells were first treated by Nocodazole for 16 h, then the drug was removed by several washes with media and a fresh media supplemented with 10 mg/ml of aphidicholine (Sigma) was applied for the next 9-10 h as it was reported for mouse embryonic stem cells (Stead et al, 2002).…”

“…Our understanding of the impact of cell cycle structure on hESCs pluripotency is far from complete (Neganova and Lako, 2008) and is mostly derived from the extension of studies of mouse embryonic stem cells, which demonstrate substantial differences in cell cycle regulation between these and somatic cells (Stead et al, 2002;Faast et al, 2004;Savatier and Malashicheva, 2004;White et al, 2005;Becker et al, 2006;Neganova and Lako, 2008).…”

“…Recent studies in hESCs suggest that these also possess an abbreviated cell cycle owing to a selective reduction of G1 phase (Becker et al, 2006), which has been attributed to the elevated mRNA levels of CYCLIN D2/cyclin-dependent kinase 4 (CDK) that are known to promote G1 progression and to very low levels of inhibitory cell cycle proteins, p21, p27 and p57 (Becker et al, 2006.…”

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…Surprisingly, several studies have recently reported that hESCs fail to fully execute the G1/S checkpoint, which may account for the fast mitogenindependent (''automated'') progression of hESCs through G1 [8][9][10].…”