Signalling pathways maintaining human embryonic stem cells

The aim of this project is to determine the function of novel genes predicted to have critical roles in preimplantation human development and in disease in later life. Due to the heterogeneity of human embryos and their precious nature, our molecular understanding of the developing embryo is limited. It is known that the period from fertilization through preimplantation development is a uniquely susceptible window in development, with the embryonic genome being activated as early as the 2cell stage (Vassena et al) and large scale epigenetic remodeling occurring immediately post-fertilization (Santos et al). Therefore, developing our understanding of preimplantation molecular mechanisms has important implications for fundamental understanding of early human development including the origins of disease (Developmental Origins of Health and Disease; DOHaD), assisted reproductive technology (ART), and therapies using human embryonic stem cells (hESCs) derived from embryos.

We have generated global gene expression microarray data and extensively mined the profiles obtained from a wide range of single embryos at developmental stages from oocyte through to blastocyst and its component parts; trophectoderm and inner cell mass (Shaw et al 2013). Our approach suggests that the MTOR/PI3K pathway, which has well documented roles in cancer and disease, also plays a crucial role in early embryo development. In addition, we have identified a number of novel and exciting genes which we believe to be important across and within different developmental stages, including epigenetic regulators. We aim to combine these findings with RNASeq, following recent success using single embryonic cells (Xue et al 2013), to provide information on novel transcripts and alternative pre-mRNA splicing.

Our laboratory as part of the North West Embryonic Stem Cell Centre (NWESCC) has generated a number of human embryonic stem cells (hESC) lines and induced pluripotent stem cells,which can form all tissues in the body and so can be used to investigate function in early developmental cell fate decisions, as well as for tissue regeneration. The Centre provides a focus for research projects on early human development, pluripotency, generation of mesodermal and endodermal differentiated derivatives and the stem cell niche..

Therefore the aims of this project are to:

Perform hESC functional studies on novel genes identified within the microarray analysis.
Perform additional microarray analysis[M1] and RNASeq on single preimplantation embryos and cells and conduct splicing analysis.

Techniques will include pre-implantation embryo culture, hESC culture, immunofluorescence, immunoprecipitation, Western blotting. Real-Time fluorescence and confocal microscopy, flow cytometry, both qualitative and quantitative RT-PCR, lentiviral mediated genetic manipulation, RNASeq and other techniques depending on the results obtained.