The role of PI3K/AKT, MAPK/ERK and NFκβ signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis

Abstract

Understanding the molecular mechanism by which pluripotency is maintained in human embryonic stem cells (hESC) is important for the development of improved methods to derive, culture and differentiate these into cells of potential therapeutic use. Large-scale transcriptional comparison of the hES-NCL1 line derived from a day 8 embryo with H1 line derived from a day 5 embryo (WiCell Inc.) showed that only 0.52% of the transcripts analysed varied significantly between the two cell lines. This is within the variability range that has been reported when hESC derived from days 5-6 embryos have been compared with each other. This implies that transcriptional differences between the cell lines are likely to reflect their genetic profile rather than the embryonic stage from which they were derived. Bioinformatic analysis of expression changes observed when these cells were induced to differentiate as embryoid bodies suggested that quite a few of the downregulated genes were components of signal transduction networks. Subsequent analysis using western blotting, flow cytometry and antibody arrays implicated components of the PI3K/AKT kinase, MAPK/ERK and NFkappabeta pathways and confirmed that these components are decreased upon differentiation. Disruption of these pathways in isolation using specific inhibitors resulted in loss of pluripotency and/or loss of viability suggesting the importance of such signalling pathways in embryonic stem cell maintenance.

Regulation of Nanog Expression by Phosphoinositide 3-Kinase-dependent Signaling in Murine Embryonic Stem Cells

“…Maintenance of murine ES cell self-renewal and proliferation are intimately linked, and PI3Ks have been implicated not only in maintenance of self-renewal of ES cells (11,31) but also in regulation of mES cell proliferation (27,29,30,44) and more recently the survival (54) and pluripotency of hES cells (33,54). Our initial report on the role of PI3Ks in self-renewal implicated enhanced activation of ERKs in this response (11), consistent with the view that the balance between STAT3 and ERK signals was key to determining ES cell fate (13).…”

“…In view of the increasing evidence for a role of PI3Ks in regulation of human ES cell fate (33,54), it will be interesting to determine whether similar mechanisms of regulation are conserved in these cells.…”

“…PI3K-mediated signaling has been implicated in an array of physiological processes, notably proliferation, cell survival, cell migration, and trafficking (25,26). As observed in many somatic cells, PI3Ks have been reported to control proliferation of mES cells (27)(28)(29)(30), whereas we (11) and others (31,32) have reported that PI3K-mediated signaling is important for maintenance of mES cell pluripotency and very recently that of hES cells (33). Here we have examined the mechanisms regulated by PI3Ks that contribute to maintenance of mES cell self-renewal.…”

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

“…For the flow cytometry analysis the hESCs were collected, processed and analyzed as described in Armstrong et al (2006) with the following antibodies: rabbit anti-cyclin D1 monoclonal antibody (FITC conjugated, clone SP4 from Abcam plc, Cambridge, UK); mouse monoclonal anti-Cyclin E (HE 12; FITC conjugated from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and R&D systems (Minneapolis, MN, USA) monoclonal anti-human/mouse SSEA-4 (APC conjugated).…”

Avaliação do Papel da Via Canônica e Não Canônica de NFB na Manutenção da Pluripotência e na Diferenciação, por Meio da Técnica de Imunoprecipitação de Cromatina

“…Deste modo, de acordo com os membros da família que estão envolvidos na ativação da via, dá-se a Interessantemente, estudos realizados anteriormente em nosso grupo mostraram por análise de expressão gênica que células-tronco hematopoéticas primitivas de cordão umbilical quando comparadas com células da melula óssea apresentaram alta expressão de fatores de transcrição relacionados com a hematopoiese primitiva e a auto-renovação, entre eles componentes chaves da via NFκB não canônica, RelB e NFΚB2, sugerindo que a via NFκB pode estar envolvida na regulação da pluripotência em células-tronco (PANEPUCCI et al, 2007). Outros estudos, muitos deles contraditórios, têm mostrado a participação dos componentes da via canônica e não-canônica de NFκB nos processos de diferenciação e destino celular ou manutenção da pluripotência (ARMSTRONG et al, 2006;KANG et al, 2007; LÜNINGSCHRÖR; KALTSCHMIDT;KALTSCHMIDT, 2012;YANG et al, 2010).…”