Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
03 02 2023
03 02 2023
Historique:
received:
19
08
2022
accepted:
31
01
2023
pubmed:
4
2
2023
medline:
8
2
2023
entrez:
3
2
2023
Statut:
epublish
Résumé
V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.
Identifiants
pubmed: 36737643
doi: 10.1038/s41598-023-29165-z
pii: 10.1038/s41598-023-29165-z
pmc: PMC9898512
doi:
Substances chimiques
Hedgehog Proteins
0
Homeobox Protein Nkx-2.2
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2008Subventions
Organisme : Wellcome Trust
ID : 108874/Z/15/Z
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/N025865/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/V002783/1
Pays : United Kingdom
Informations de copyright
© 2023. The Author(s).
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