Screening for regulators of human embryonic axis elongation in vitro
Lead Research Organisation:
University of Sheffield
Department Name: Biomedical Science
Abstract
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.
Technical Summary
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.
Planned Impact
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.
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.
Organisations
- University of Sheffield (Lead Research Organisation)
- University College London (Collaboration)
- University of Luxembourg (Collaboration)
- Erasmus University Rotterdam (Collaboration)
- Eindhoven University of Technology (Collaboration)
- University of Leuven (Collaboration)
- Aalto University (Collaboration)
People |
ORCID iD |
Anestis Tsakiridis (Principal Investigator) |
Publications
Wymeersch FJ
(2021)
Understanding axial progenitor biology in vivo and in vitro.
in Development (Cambridge, England)
Cooper F
(2022)
Towards clinical applications of in vitro-derived axial progenitors.
in Developmental biology
Cooper F
(2022)
Shaping axial identity during human pluripotent stem cell differentiation to neural crest cells.
in Biochemical Society transactions
Frith TJR
(2020)
Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In-Vitro-Derived Neural Crest.
in Stem cell reports
Cooper F
(2024)
Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors.
in Development (Cambridge, England)
Wind M
(2021)
In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells
in Current Protocols
Frith TJ
(2018)
Human axial progenitors generate trunk neural crest cells in vitro.
in eLife
Frith T
(2018)
Human axial progenitors generate trunk neural crest cells
Description | Part of the grant involved understanding how a specific progenitor population of cells helps the building of the spinal cord and vertebral column of human embryos/fetuses by producing their precursor cell types. We have now developed "recipes" for growing these cell types (progenitors and spinal cord/vertebral column precursors) efficiently in the petri dish and we discovered that we can also generate a third cell type (called trunk neural crest) from these same progenitor cells. Trunk neural crest are a very important cell population as they give rise to neurons that innervate crucial organs such as the heart as well as specialised neuroendocrine cell types. This work has now been published in elife (open access: https://elifesciences.org/articles/35786). We have also defined the signals required for generating another important progenitor cell population that gives rise to the enteric nervous system and we have submitted these findings have now been published (https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(20)30301-5). We now hope to utilize these progenitors, as part of a new MRC--funded project, for developing the preclinical basis for a cell therapy aiming to treat a congenital disease called Hirschsprung disease. |
Exploitation Route | Our new findings will be of great interest to researchers working in both developmental biology and stem cells as well as clinical geneticists/clinicians trying to understand the molecular basis of some forms of spina bifida/vertebral birth defects, entetric neuropathies (E.g. Hirschsprung disease) as well as neuroblastoma which is the most common paediatric solid tumour and arises in trunk neural crest cells and their early derivatives. Thus they are also likely to be of interest to related patient groups and the biotech/drug development sector. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
URL | https://www.sheffield.ac.uk/bms/feeds/organ-function-cells-early-human-embryonic-development-1.798382 |
Description | BBSRC White Rose DTP 4-year PhD studentship. "How do enhancers control the expression of the same transcriptional factor in different regions of the nervous system?" |
Amount | £90,608 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2020 |
End | 05/2024 |
Description | Establishment of an in vitro model of neuroblastoma initiation using pluripotent stem cell differentiation |
Amount | £72,656 (GBP) |
Organisation | Children's Cancer and Leukaemia Group (CCLG) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2019 |
End | 02/2021 |
Description | Horizon 2020 - Research and Innovation Framework Programme. Connecting neural networks: Nervous-system-on-Chip Technology. |
Amount | € 6,807,867 (EUR) |
Funding ID | Grant agreement ID: 824070 |
Organisation | European Commission H2020 |
Sector | Public |
Country | Belgium |
Start | 01/2019 |
End | 12/2023 |
Description | MRC research grant: Developing a human pluripotent stem cell-based strategy for treating Hirschsprung disease |
Amount | £1,251,179 (GBP) |
Funding ID | MR/V002163/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2021 |
End | 12/2023 |
Title | Generation of vagal and trunk neural crest cells |
Description | The grant facilitated the development of protocols for the efficient in vitro generation of vagal and trunk neural crest cells and their derivatives (enteric neurons/glia and sympathoadrenal lineages respectively) from human pluripotent stem cells. |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Our protocol for generating vagal neural crest cells and their derivatives comprises the basis for the further development of a cell therapy approach for treating Hirschsprung disease, which is the focus of a separate subsequent MRC-funded grant currently in progress. Our protocol for generating trunk neural crest cells can be utilised for the in vitro modelling of birth defects and cancers arising in these cells such as neuroblastoma. |
URL | https://elifesciences.org/articles/35786 |
Title | In vitro generation of human posterior spinal cord cells |
Description | The grant facilitated the development of a protocol for the efficient in vitro generation of posterior spinal cord cells and motor neurons from human pluripotent stem cells. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | The cells generated using this protocol can be potentially utilised for cell therapy and disease modelling application e.g for the treatment of spinal cord injuries and motor neuron disease respectively. |
Title | Axial progenitors generate trunk neural crest cells in vitro-Expression profiling by array |
Description | Gene expression profiling utilised total RNA extracted from ES cells (N=3); hPSC derived Cranial neural crest precursors (N=3); hPSC derived Cranial neural crest cells (N=3); hPSC derived Cranial neural crest cells after RA treatment to posteriorise (N=3); hPSC derived Neuromesodermal progenitors (N=3); hPSC derived Trunk neural crest progenitors (N=3); hPSC derived trunk neural crest cells (N=3) |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Gene expression profiling of regionally distinct hES-derived neural crest cell populations would be a useful resource for the wider scientific community, in particular clinical geneticist and developmental biologists interested in neurocristopathies. |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109267 |
Title | Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives |
Description | RNA sequencing analysis of in vitro-derived early spinal cord progenitors |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Data can be used as a resource by the wider scientific community (Especially developmental biologists and neuroscientists) to understand human spinal cord development. |
Description | Collaboration with researchers and clinicians from UCL Great Ormond Street Institute of Child Health on developing a stem cell therapy against Hirschsprung disease |
Organisation | University College London |
Department | Great Ormond Street Institute of Child Health |
Country | United Kingdom |
Sector | Public |
PI Contribution | Intellectual/technical expertise/support in terms of generating hPSC-derived enteric neuron progenitors |
Collaborator Contribution | Intellectual/technical expertise/support in terms of functional characterisation of in vitro derived enteric neuron progenitors |
Impact | Multidisciplinary collaboration involving basic scientists and clinicians Publication: DOI:https://doi.org/10.1016/j.stemcr.2020.07.024 Follow-up funding: Work also led to a successful joint grant application to the MRC |
Start Year | 2017 |
Description | Participation in H2020-funded consortium |
Organisation | Aalto University |
Country | Finland |
Sector | Academic/University |
PI Contribution | We provided intellectual and technical support in terms of generating hES-cell derived cell populations |
Collaborator Contribution | Access to facilities, reagent provision, intellectual input, training. |
Impact | Multidisciplinary collaboration involving stem cell biologists, neuroscientists and engineers. |
Start Year | 2019 |
Description | Participation in H2020-funded consortium |
Organisation | Eindhoven University of Technology |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We provided intellectual and technical support in terms of generating hES-cell derived cell populations |
Collaborator Contribution | Access to facilities, reagent provision, intellectual input, training. |
Impact | Multidisciplinary collaboration involving stem cell biologists, neuroscientists and engineers. |
Start Year | 2019 |
Description | Participation in H2020-funded consortium |
Organisation | Erasmus University Rotterdam |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We provided intellectual and technical support in terms of generating hES-cell derived cell populations |
Collaborator Contribution | Access to facilities, reagent provision, intellectual input, training. |
Impact | Multidisciplinary collaboration involving stem cell biologists, neuroscientists and engineers. |
Start Year | 2019 |
Description | Participation in H2020-funded consortium |
Organisation | University of Leuven |
Country | Belgium |
Sector | Academic/University |
PI Contribution | We provided intellectual and technical support in terms of generating hES-cell derived cell populations |
Collaborator Contribution | Access to facilities, reagent provision, intellectual input, training. |
Impact | Multidisciplinary collaboration involving stem cell biologists, neuroscientists and engineers. |
Start Year | 2019 |
Description | Participation in H2020-funded consortium |
Organisation | University of Luxembourg |
Country | Luxembourg |
Sector | Academic/University |
PI Contribution | We provided intellectual and technical support in terms of generating hES-cell derived cell populations |
Collaborator Contribution | Access to facilities, reagent provision, intellectual input, training. |
Impact | Multidisciplinary collaboration involving stem cell biologists, neuroscientists and engineers. |
Start Year | 2019 |
Description | Interview on research to Children's Cancer and Leukaemia Group (CCLG) |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | Interview on lab's research as part of a series of Research Focus interviews by the childhood cancer charity Children's Cancer and Leukaemia Group (CCLG). |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.cclg.org.uk/news/research-focus-dr-anestis-tsakiridis |
Description | University press release related to eLife publication |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Not known |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.sheffield.ac.uk/bms/feeds/organ-function-cells-early-human-embryonic-development-1.79838... |