A CELL ATLAS OF THE DEVELOPING HUMAN SENSORY NERVOUS SYSTEM: INVESTIGATING DEVELOPMENT TO UNDERSTAND DISEASE

Context of the research:
Pain is the main reason that diseases of the muscles and joints lead to disability. In particular, for the tens of millions of individuals who suffer from Osteoarthritis (OA) , pain can be debilitating at end stages and be present even at rest, preventing the use of joints in the hand, hip and knees. Around the world, this tremendous societal and monetary burden is set to rise alongside an aging population. Currently, there is no cure for osteoarthritis and end stage disease is treated with joint replacement, a costly and major operation. Importantly, we lack effective pain-relief medications for OA.

Until recently, it was thought that wear-and-tear of the protective lining of the joint, and increased bone-to-bone contact is what leads to pain in disease. Interestingly, recent studies from various research groups have shown that new nerves sprout into the joint surface as part of the disease process of OA. These nerves appear to be present in individuals with painful OA, but is absent in those with less painful OA. In adulthood, nerves normally have limited capacity to grow, so this leads us to think that disease nerves are driven by processes similar to those that are active when we our nervous system begins to develop while an embryo in the womb.

The objective of this project is therefore to apply cutting edge technology to investigate the development of the human sensory nervous, comparing it to what happens in disease, with the aim of identifying novel ways of interfering with it, in order to treat pain.

Aims and objectives:
First, we aim to understand how the sensory nervous system forms in the developing human. To achieve this, we will use single-cell sequencing technologies. This will allow us to detect what drives different types of developing nerves, one by one. We aim to form a classification system for these nerves, sorting them by their function, for example, some nerves may sense touch and others may sense pain, and they might be driven by different signals to grow.
Secondly, we will map the distribution of these developing nerves in space, using a type of technology known as spatial transcriptomics. This will form an atlas of the nerves throughout the body and allow us to understand how nerves are queued to grow towards their destination during development.

Then, we will map nerves in the diseased human osteoarthritic knee joint in adulthood. We will apply innovative technologies to first identify areas where nerves are present, and then sequence the genes activated within these areas. This will allow us to understand whether nerves growing in disease are similar or different to nerves that grow during development.Lastly, we will use computational programs to compare developmental nerves to disease-nerves. This will allow us to decide how to target the disease-nerves.

Potential applications and benefits:
We will be the first group to perform single-cell sequencing on the developing nervous system in human. This will allow discovery of new cell-types, and create an atlas for them throughout the human body.
These findings will further our understanding of all painful conditions. In the case of osteoarthritis, it may allow us to identify targets that can be exploited to allow depletion of the pain-associated nerves described above. We aim to disseminate our findings to allow public access by the research community. This will lead to improved understanding of other diseases, such as in cancer, where nerves can also sprout during the disease process.

In the long term, our findings can also be applied to help improve models of nerves grown in the laboratory, and potentially also inform ways of promoting nerves to regenerate in the body.

I aim to test the hypothesis that the process of neo-innervation of the adult osteoarthritic joint by pain-associated nerve afferents recapitulates transcriptional profiles that are active in the developing human fetal sensory nervous system, with a view to obtain mechanistic insight, and identify potential therapeutic targets for pain.

First, I will create the first human cell atlas (HCA) of the developing sensory nervous system. Secondly, I will identify the transcriptional profiles of adult osteoarthritic joint cartilage that is innervated by pathogenic nociceptive afferents (absent in the osteochondral junction in health). Finally, I will compare the transcriptional profiles in these states to test our hypothesis, and additionally prioritise potential treatment targets.

Single-cell RNA sequencing(scRNAseq) will be applied to human dorsal root ganglia samples between the fetal ages of 6 to 20 weeks, with 1-2 sample(s) each gestational week.
To create a spatial atlas, single-nuclei RNAseq and Visium RNAseq (10X genomics) will be applied to sequential slices of the same tissue sample at ages of 6-7 weeks and 18 weeks. In adult osteoarthritis tissue, multiplex in situ hybridization will be used to identify regions of interest by neuronal markers followed by the use of Nanostring GeoMx for targeted sequencing. Finally, computational integration will be applied to reveal reciprocal matches in cell states between development and disease. Potential treatment targets will be tested in further projects through in vitro and in vivo experiments.

The study will create the first HCA of the developing nervous system and transcriptional profiles of disease, the findings will provide new insights into normal human development as well as disease, with therapeutic implications. The developmental dataset can also be studied to understand nerve regeneration in adulthood, with implications in regenerative medicine, and in generating in vitro models of sensory nerves.