A simplified method for generating Purkinje cells from human-induced pluripotent stem cells
The establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes f...
| Main Authors: | , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Springer US
2018
|
| _version_ | 1826268436465975296 |
|---|---|
| author | Watson, L Wong, M Vowles, J Cowley, S Becker, E |
| author_facet | Watson, L Wong, M Vowles, J Cowley, S Becker, E |
| author_sort | Watson, L |
| collection | OXFORD |
| description | The establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes for study in vitro. However, to date, only a handful of studies have generated Purkinje cells from human pluripotent stem cells, with most of these protocols proving challenging to reproduce. Here, we describe a simplified method for the reproducible generation of Purkinje cells from human iPSCs. After 21 days of treatment with factors selected to mimic the self-inductive properties of the isthmic organiser-insulin, fibroblast growth factor 2 (FGF2), and the transforming growth factor β (TGFβ)-receptor blocker SB431542-hiPSCs could be induced to form En1-positive cerebellar progenitors at efficiencies of up to 90%. By day 35 of differentiation, subpopulations of cells representative of the two cerebellar germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more accessible, in order to enable future advances in the field. |
| first_indexed | 2024-03-06T21:09:38Z |
| format | Journal article |
| id | oxford-uuid:3db1bb6c-bbcd-4613-a610-4aa99d0cbfa0 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:09:38Z |
| publishDate | 2018 |
| publisher | Springer US |
| record_format | dspace |
| spelling | oxford-uuid:3db1bb6c-bbcd-4613-a610-4aa99d0cbfa02022-03-26T14:20:59ZA simplified method for generating Purkinje cells from human-induced pluripotent stem cellsJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:3db1bb6c-bbcd-4613-a610-4aa99d0cbfa0EnglishSymplectic Elements at OxfordSpringer US2018Watson, LWong, MVowles, JCowley, SBecker, EThe establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes for study in vitro. However, to date, only a handful of studies have generated Purkinje cells from human pluripotent stem cells, with most of these protocols proving challenging to reproduce. Here, we describe a simplified method for the reproducible generation of Purkinje cells from human iPSCs. After 21 days of treatment with factors selected to mimic the self-inductive properties of the isthmic organiser-insulin, fibroblast growth factor 2 (FGF2), and the transforming growth factor β (TGFβ)-receptor blocker SB431542-hiPSCs could be induced to form En1-positive cerebellar progenitors at efficiencies of up to 90%. By day 35 of differentiation, subpopulations of cells representative of the two cerebellar germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more accessible, in order to enable future advances in the field. |
| spellingShingle | Watson, L Wong, M Vowles, J Cowley, S Becker, E A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title | A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title_full | A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title_fullStr | A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title_full_unstemmed | A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title_short | A simplified method for generating Purkinje cells from human-induced pluripotent stem cells |
| title_sort | simplified method for generating purkinje cells from human induced pluripotent stem cells |
| work_keys_str_mv | AT watsonl asimplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT wongm asimplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT vowlesj asimplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT cowleys asimplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT beckere asimplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT watsonl simplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT wongm simplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT vowlesj simplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT cowleys simplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells AT beckere simplifiedmethodforgeneratingpurkinjecellsfromhumaninducedpluripotentstemcells |