Heterogeneity and cellular population dynamics in the placenta

Context
Placenta-related complications of pregnancy are a major challenge for women's health. Stillbirth is a major cause of perinatal death, causing more than 3,000 deaths each year in the UK and ~2.6million worldwide. Fetal growth restriction results in short term morbidity, poor educational outcomes and predisposes to diabetes and cardiovascular disease in later life. Mechanistic understanding and effective therapies for these complications are lacking. The work proposed here will provide a detailed atlas of all the different cell types in the placenta -currently we don't know how many different cell types and sub-types there are. We will also determine which RNAs are present in each of these cells and this is directly relevant to cell function. Knowledge of how the different cell types arise is key to understanding normal placental development. Methods for the analysis of the mRNA within single cells have recently been developed. However, as the barrier between maternal blood and fetal tissue is a multi-nucleated syncytium current single "cell" RNA-sequencing methods are inappropriate and new approaches are needed. This proposal describes such new methods and their application to the placenta.

Aims
The overarching aim of this work is to use single cell RNA sequencing to characterise placental cellular heterogeneity in mouse and man and to define regulatory steps in trophoblast differentiation. To address this aim we will answer the following 3 specific questions:

1) How many cell types are there in the placenta and what are their distinguishing features? This will necessitate the development of new methods, specifically to allow the analysis of individual nuclei and the analysis of non-coding RNA in many thousands of single nuclei.

2) How does the placental cell population change over time? We will evaluate the different cell populations by single nucleus sequencing from tissue collected from 5 human placentas in the first trimester and also at term. We will compare these with data from samples of mouse placenta collected at 5 different times in pregnancy.

3) What are the mechanisms that regulate this heterogeneity?
Murine trophoblast stems cells can be grown under conditions that induce differentiation. We will sequence single cells from multiple time points during differentiation. Sampling a population of differentiating cells at the single cell level allows identification of the intermediate populations during differentiation. We will identify regulatory factors at the branch points in this process and test whether over expression or knocking out these genes alters the differentiation.

Potential applications and benefits
Preventing even a small proportion of still births and other complications of pregnancy would be a significant benefit. Good obstetric care means that many pregnancies complicated by poor placental development end in live births. However, babies whose intrauterine life was compromised are at risk for long-term chronic diseases, such as cardiovascular disease and diabetes. Better understanding of placental growth and function has the potential to improve pregnancy outcome and this will have both short and long term health and economic benefits. These are distant benefits but more immediate gains will be in the fields of placental, developmental, gene regulatory biology. The methods proposed are innovative and require cross-disciplinary work (microfluidics, chemistry, molecular biology and bioinformatics) and so will benefit all these disciplines.

The work proposed here is to be carried out with a collaborative industrial partner - Sphere Fluidics Limited. Close collaboration between the University, the Babraham Institute and Sphere Fluidics will further enhance interactions among biotech/pharma and academia around Cambridge. This will be a benefit to these three parties and may attract R&D investment and increase economic activity to the benefit of the UK economy.

Placenta-related complications of pregnancy are a major challenge for women's health. Stillbirth is a major cause of perinatal death, causing more than 3,000 deaths each year in the UK and ~2.6million worldwide.
The overarching aim of this work is to use single cell RNA sequencing to characterise placental cellular heterogeneity in mouse and man and to define regulatory steps in trophoblast differentiation. Specifically:

1) How many cell types are there in the placenta and what are their distinguishing features?
Single cell RNA-Seq has the potential to characterise cellular diversity and provide information on the functional repertoire of individual cells. However, the barrier between maternal blood and fetal tissue is a multi-nucleated syncytium. We will improve our existing Drop-Seq methods for the analysis of nuclei. Expertise in microfluidics is provided by the Abell laboratory and Sphere Fluidics Ltd. We will develop methods for sequencing noncoding RNA from cells and nuclei. Key to this is trapping a nucleus in a gel-droplet and capturing liberated RNA on the hydrogel matrix. The gel-droplets will be collected and processed in an aqueous environment. This innovative approach allows buffer exchange and enzymatic steps to be performed under optimum conditions.

2) How does the placental cell population change over time?
We will sequence the total RNA from ~15,000 single nuclei from 5 placentas collected in the first trimester and at term. We will compare these with similar data from mouse pregnancy (sampled at E7.5, E8.5, E9.5, E14.5 & E17.5).

3) What are the mechanisms that regulate this heterogeneity?
We will use mouse TSCs to model trophoblast differentiation. We will use the concept of pseudo-time to unravel individual steps and identify intermediate populations during differentiation into the specific cell types. Regulatory factors (not just transcription factors) will be manipulated by over-expression or CRISPR/Cas9 knock out in murine TSCs.

Who will benefit from this research?

In the long term this work will contribute to an improvement in global human health and wellbeing through a reduction in complications of pregnancy. In the UK there were more than 3,000 stillbirths in 2015, while worldwide 14 million infants are born growth restricted and 50,000 women die from pre-eclampsia. Preventing even a small proportion of these complications would therefore have major health, societal and economic benefits. Good obstetric care means that many pregnancies complicated by poor placental development end in live births. However, babies whose intrauterine life was compromised are at risk for long-term chronic diseases, such as cardiovascular disease and diabetes. Hence the economic and societal impact of poor placental development is considerable.

How might they benefit from this research?

Scientists and clinicians investigating the mechanism underlying placental growth and function will benefit directly through new knowledge arising from this research. The University of Cambridge has a very high density of researchers working on the placenta and is home to the philanthropically endowed Centre for Trophoblast research (CTR, http://www.trophoblast.cam.ac.uk). This award would extend this emerging technology within the centre (and more widely). This would enhance research capacity. Furthermore, we believe we will be the first group in the world seeking to perform placental single cell sequencing on this scale. Therefore, the resulting data will be unique and will enhance the UK's scientific reputation for high quality research.

This application is for an Industrial Partnership Award (IPA) and the work is to be carried out with a collaborative partner - Sphere Fluidics Limited. We anticipate this work may generate new IP (new methods, systems of analysis and possibly identification of cellular control mechanisms) that could be exploited leading to new wealth and job creation. As this is an application for an IPA, Sphere Fluidics would be an excellent partner for this. Potentially they and other biotech and pharma companies may benefit. Close collaboration between the University, the Babraham Institute and Sphere Fluidics will further enhance interactions among biotech/pharma and academia around Cambridge. This should attract R&D investment and increase economic activity. It is relevant that two of the investigators (DSC-J and CA) have previously founded successful biotech companies and filed and licenced multiple patents.

More generally the work will enhance the knowledge of economy and promote the health of a variety of scientific disciplines. The work is innovative, cross disciplinary and at the forefront of biomedical science, so will enhance the UKs scientific reputation.