Stem cell models of the trophoblast

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How do we sexually reproduce? Compared to other areas of human and animal biology the biology of reproduction is less investigated. Despite its essential function of keeping the fetus alive until birth, the placenta is the least studied organ in the body. Searching for research articles about the “placenta” finds 90,909 publications, while the kidney has over 800,000 and brain over 1,000,000. There are many reasons for this lack of placental research. First, failures of the placenta's growth or function often lead to stillbirth or miscarriage, meaning there is no patient to treat (aside from the grieving parents). Second is that female-specific biology has been traditionally understudied. ***We have serious medical issues that we cannot solve. The most common pathologies of pregnancy, such as the hypertensive disorder of preeclampsia and preterm birth (combined affecting nearly 10% of all pregnancies) have no treatments, other than delivery of the baby and transfer to intensive care. This failure is due to the first problem; we have not conducted sufficient basic science research to understand this most critical organ to mammalian reproduction and development. ***Advancing the basic biology of the trophoblast. Within my proposed research, my group will assess human and mouse placenta tissues to characterise the diversity of cell types and their changes during development. To accomplish this, we will use single-cell gene expression methods (RNA-sequencing) and statistical clustering. Additionally, new bioinformatics methods that utilise the ratio of spliced to non-spliced RNA are used to identify developmental connectivity between these clusters of cells. Stem cell models of mouse and human trophoblast will be used to characterise cell signalling and expressed genes essential to trophoblast development. Signalling is addressed using activators and inhibitors to guide cells into specific mature trophoblast cell types. Gene function is investigated using gene deletion by the CRISPR-CAS9 method. In particular, I will focus on imprinted genes. ***Imprinting and the trophoblast. Imprinting is a process where a gene's expression is differentially regulated depending on its parent of origin. The evolution of gene imprinting occurred with the evolution of the trophoblast and the placenta. Imprinting is typically controlled by differential DNA methylation, which we will observe this by DNA sequencing. Many known imprinted genes are found in sets associated with one or more long non-coding RNAs. RNA sequencing can identify imprinted genes as genes that are expressed by only one allele based on the presence or absence of known sequence polymorphisms between a genes alleles. Through the integrated assessment of DNA methylation, RNA-sequencing and genomic sequences I will identify novel and human-specific imprinted genes for deletion to assess their function on trophoblast cell development. **