Investigating the role of cell cycle regulators in mesoderm specification

Mesoderm is one of the three primary germ layers from which the cardiovascular system, muscle and bone originate and derivatives of the mesoderm lineage are affected in a number of pathologies. Therefore, understanding the mechanisms regulating formation of mesoderm is interesting for a diversity of diseases and clinical application. In vivo study of human development beyond gastrulation is technically challenging and the mechanisms controlling mesoderm specification are difficult to study since the maximum number of days allowed to grow human embryos is 14 days. Thus, in this dissertation I use human pluripotent stem cells (hPSCs) as a simplified model of human development. Studies have shown that the cell cycle machinery plays a direct role in the differentiation of endoderm and ectoderm lineages but its role in guiding mesoderm subtype formation remains elusive. In this dissertation, I provide new insights of the importance of the cell cycle regulators in mesoderm specification. I first developed tools such as the FUCCI2A reporter line to isolate cells in the different cell cycle phases and to investigate propensity of mesoderm differentiation. I have shown that the propensity of differentiation into the three mesoderm subtypes lateral plate mesoderm, cardiac mesoderm and presomitic mesoderm varies during the cell cycle phases, with differentiation being more efficient in the G1 and to a lesser extend in G2/M phase. Furthermore, I developed a protocol where cells can be efficiently synchronised in the different cell cycle phases using the G2/M inhibitor nocodazole. Using this tool, I showed that developmental signalling pathways such as BMP and WNT are active in all cell cycle phases indicating that alternative mechanisms are involved in the differentiation process. In order to further explore these mechanisms, I investigated the role of cell cycle regulators controlling the G1 and G2 checkpoint. I have shown that the cell cycle regulators CDK4/6, CDK2, Retinoblastoma phosphorylation and CDK1 are essential for mesoderm subtype formation. Furthermore, I have shown that CDK1 regulates the activity of ERK1/2 signalling, an important pathway for the differentiation process confirming the existence of complex interplays between cell cycle machinery, signalling pathways and transcription factors in mesoderm subtype formation. This knowledge will be useful to further improve protocols for generating mesoderm subtypes from hPSCs for clinical applications such as drug screening, disease modelling and cell based therapy.