Screening for regulators of human embryonic axis elongation in vitro

Directing pluripotent stem cells (PSCs) to generate ("differentiate") various cell types in the petri dish is an attractive tool for understanding how embryos develop and a promising route towards therapies. However, our ability to produce cell types corresponding to the lower spinal cord (the thoracic, lumbar and sacral regions) and skeletal muscle is currently very limited. This is because conventional PSC differentiation methods fail to produce the common embryonic precursor of these cell types known as neuromesodermal progenitors (NMPs). We have recently succeeded in devising a protocol for converting efficiently human PSCs into NMPs (hNMPs). These in vitro generated NMPs appear to be a promising starting material for producing lower spinal cord and skeletal muscle cells. However, we still do not know much about the signals and the molecular mechanisms driving the generation of these cell types from hNMPs. The experiments proposed here will test various chemicals in order to identify the best "recipes" for pushing human NMPs to become exclusively either lower spinal cord or skeletal muscle cells. We will also examine how the binding of certain proteins known as transcription factors to the DNA of NMPs influences their decision to remain NMPs or differentiate into spinal cord/skeletal muscle cells. This work will lead to a better understanding of NMP biology and pave the way for the use of their differentiation products in the clinic.

The differentiation of human pluripotent stem cells (hPSCs) is a powerful tool for both dissecting embryonic development and producing clinically relevant cell populations in vitro. However, current hPSC differentiation protocols cannot generate efficiently certain important cell lineages such as posterior neurectoderm (PNE) and paraxial mesoderm (PXM) which give rise to thoracic/lumbosacral spinal cord and trunk skeletal muscle respectively. This limitation is due to the inability of conventional approaches to promote the induction of neuromesodermal progenitors (NMPs), the bipotent stem cell population driving axis elongation through the production of PNE and PXM in vertebrate embryos. We have recently described the efficient induction of NMPs from hPSCs (hNMPs) and our preliminary data indicate that hNMPs can be utilized as the ideal starting cell population for generating "refractory" PNE and PXM derivatives. However, the optimal conditions for the homogeneous differentiation of hNMPs into either PNE or PXM remain to be determined. Furthermore, it is currently impossible to maintain and propagate pure cultures of hNMPs. This proposal aims to address these issues by precisely defining the molecular hallmarks of NM bipotency and PNE/PXM specification. Specifically, we will test the effect of manipulating various signalling pathways, found by whole transcriptome analysis to be active in hNMP cultures, on hNMP maintenance/differentiation using high content imaging. Furthermore, we will decipher the binding targets of Brachyury and FOXB1, two transcription factors initiating PXM and PNE specification respectively, in order to both identify regulators directing the transition of hNMPs into these lineages.

The following groups of people will benefit from the proposed research project:

1) Staff employed on the project- specialist and generic/transferable skills training
The postdoc employed on this project will receive extensive technical training in human pluripotent stem cell (hPSC) culture and differentiation. Our collaboration with Dr Abdenour Sufi who is an expert in the biochemical analysis of pioneer transcription factors, chromatin biology and sequencing techniques will mediate the transfer of these skills to the personnel involved in the project. This knowledge transfer will be mediated by visits to Dr Sufi's lab in Edinburgh.

2) Industry
The potential to generate homogeneously large numbers of thoracic/lumbosacral spinal cord cell types and skeletal muscle from hPSCs and induced pluripotent (iPS) stem cells will open up new avenues towards drug discovery e.g. through lead compound screening using iPS cells from both healthy individuals and patients suffering from degenerative conditions such as Amyotrophic lateral sclerosis.

3) Patients
The proposed project aims to facilitate the in vitro derivation of cell populations such as lumbosacral spinal cord neurons/glia and skeletal muscle. It is thus likely to benefit, from both a translational and basic biology perspective, patients affected from devastating degenerating conditions affecting these cell types e.g. motor neuron disease, spinal muscular atrophy and Duchenne muscular dystrophy.