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TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation

The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson’s disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neura...

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Autores principales: Sibuea, Shanti, Ho, Joan K., Pouton, Colin W., Haynes, John M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9928866/
https://www.ncbi.nlm.nih.gov/pubmed/36819101
http://dx.doi.org/10.3389/fcell.2023.1111705
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author Sibuea, Shanti
Ho, Joan K.
Pouton, Colin W.
Haynes, John M.
author_facet Sibuea, Shanti
Ho, Joan K.
Pouton, Colin W.
Haynes, John M.
author_sort Sibuea, Shanti
collection PubMed
description The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson’s disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neural induction followed by hedgehog signalling and an elevation of β-catenin to drive dopaminergic differentiation. Post-patterning, differentiating dopaminergic neuron cultures are permitted time for maturation after which the success of these differentiation paradigms is usually defined by expression of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. However, during maturation, culture media is often supplemented with additives to promote neuron survival and or promote cell differentiation. These additives include dibutyryl cyclic adenosine monophosphate (dbcAMP), transforming growth factor β3 (TGFβ3) and or the γ-secretase inhibitor (DAPT). While these factors are routinely added to cultures, their impact upon pluripotent stem cell-derived mDA phenotype is largely unclear. In this study, we differentiate pluripotent stem cells toward a dopaminergic phenotype and investigate how the omission of dbcAMP, TGFβ3 or DAPT, late in maturation, affects the regulation of multiple dopaminergic neuron phenotype markers. We now show that the removal of dbcAMP or TGFβ3 significantly and distinctly impacts multiple markers of the mDA phenotype (FOXA2, EN1, EN2, FOXA2, SOX6), while commonly increasing both MSX2 and NEUROD1 and reducing expression of both tyrosine hydroxylase and WNT5A. Removing DAPT significantly impacted MSX2, OTX2, EN1, and KCNJ6. In the absence of any stressful stimuli, we suggest that these culture additives should be viewed as mDA phenotype-modifying, rather than neuroprotective. We also suggest that their addition to cultures is likely to confound the interpretation of both transplantation and disease modelling studies.
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spelling pubmed-99288662023-02-16 TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation Sibuea, Shanti Ho, Joan K. Pouton, Colin W. Haynes, John M. Front Cell Dev Biol Cell and Developmental Biology The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson’s disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neural induction followed by hedgehog signalling and an elevation of β-catenin to drive dopaminergic differentiation. Post-patterning, differentiating dopaminergic neuron cultures are permitted time for maturation after which the success of these differentiation paradigms is usually defined by expression of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. However, during maturation, culture media is often supplemented with additives to promote neuron survival and or promote cell differentiation. These additives include dibutyryl cyclic adenosine monophosphate (dbcAMP), transforming growth factor β3 (TGFβ3) and or the γ-secretase inhibitor (DAPT). While these factors are routinely added to cultures, their impact upon pluripotent stem cell-derived mDA phenotype is largely unclear. In this study, we differentiate pluripotent stem cells toward a dopaminergic phenotype and investigate how the omission of dbcAMP, TGFβ3 or DAPT, late in maturation, affects the regulation of multiple dopaminergic neuron phenotype markers. We now show that the removal of dbcAMP or TGFβ3 significantly and distinctly impacts multiple markers of the mDA phenotype (FOXA2, EN1, EN2, FOXA2, SOX6), while commonly increasing both MSX2 and NEUROD1 and reducing expression of both tyrosine hydroxylase and WNT5A. Removing DAPT significantly impacted MSX2, OTX2, EN1, and KCNJ6. In the absence of any stressful stimuli, we suggest that these culture additives should be viewed as mDA phenotype-modifying, rather than neuroprotective. We also suggest that their addition to cultures is likely to confound the interpretation of both transplantation and disease modelling studies. Frontiers Media S.A. 2023-02-01 /pmc/articles/PMC9928866/ /pubmed/36819101 http://dx.doi.org/10.3389/fcell.2023.1111705 Text en Copyright © 2023 Sibuea, Ho, Pouton and Haynes. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Sibuea, Shanti
Ho, Joan K.
Pouton, Colin W.
Haynes, John M.
TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title_full TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title_fullStr TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title_full_unstemmed TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title_short TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
title_sort tgfβ3, dibutyryl camp and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9928866/
https://www.ncbi.nlm.nih.gov/pubmed/36819101
http://dx.doi.org/10.3389/fcell.2023.1111705
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