A New Selection Strategy to Enrich Dopaminergic Neurons Facilitates Comparisons Between Patient iPSC-Derived Lines
6 affiliations
- 1Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
- 3Core Facilities, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
- 4Nash Family Department of Neuroscience at Mount Sinai, New York, NY 10029, USA
- 5Center for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
- 6DataTecnica LLC., Washington, DC 20037, USA
Abstract
Comparative studies of dopaminergic (DA) neurons differentiated from induced pluripotent stem cells (iPSCs) derived from patients discordant for parkinsonism, especially siblings, provide a valuable model to explore factors underlying Parkinson disease (PD) pathogenesis. However, such studies are complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich DA neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene, placing its expression under the endogenous promoter. Using iPSC-derived neurons from a healthy individual (HT711), we achieved selective enrichment of TH+ neurons through geneticin selection without compromising differentiation capacity, as demonstrated by Western blots, immunocytochemistry and flow cytometry. We then applied the method to iPSCs derived from two siblings of HT711, one with Gaucher disease (GD) alone and one with both PD and GD. Quantification of multiple DA neuron markers through whole-cell proteomics confirmed their comparable enrichment in the three lines, demonstrating that the new method can be applied to iPSC lines from different individuals to standardize DA neuron cultures. While we focus on methodological developments here, a companion analysis of the same whole-cell proteomics dataset, examining molecular features potentially associated with disease statuses, has been reported separately.
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BibTeX
@article{hertz2026selection,
title = {{A New Selection Strategy to Enrich Dopaminergic Neurons Facilitates Comparisons Between Patient iPSC-Derived Lines}},
author = {Ellen Hertz and Gani Perez and Krystyna Rytel and Martha Kirby and Stacie Anderson and Stephen Wincovitch and Tim Ahfeldt and Joel Blanchard and Ying Hao and Ziyi Li and Kate Andersh and Yue Andy Qi and Ellen Sidransky and Yu Chen},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27219394},
url = {https://www.mdpi.com/1422-0067/27/21/9394},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A New Selection Strategy to Enrich Dopaminergic Neurons Facilitates Comparisons Between Patient iPSC-Derived Lines
AU - Ellen Hertz
AU - Gani Perez
AU - Krystyna Rytel
AU - Martha Kirby
AU - Stacie Anderson
AU - Stephen Wincovitch
AU - Tim Ahfeldt
AU - Joel Blanchard
AU - Ying Hao
AU - Ziyi Li
AU - Kate Andersh
AU - Yue Andy Qi
AU - Ellen Sidransky
AU - Yu Chen
PY - 2026
DA - 2026/10/22
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219394
UR - https://www.mdpi.com/1422-0067/27/21/9394
AB - Comparative studies of dopaminergic (DA) neurons differentiated from induced pluripotent stem cells (iPSCs) derived from patients discordant for parkinsonism, especially siblings, provide a valuable model to explore factors underlying Parkinson disease (PD) pathogenesis. However, such studies are complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich DA neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene, placing its expression under the endogenous promoter. Using iPSC-derived neurons from a healthy individual (HT711), we achieved selective enrichment of TH+ neurons through geneticin selection without compromising differentiation capacity, as demonstrated by Western blots, immunocytochemistry and flow cytometry. We then applied the method to iPSCs derived from two siblings of HT711, one with Gaucher disease (GD) alone and one with both PD and GD. Quantification of multiple DA neuron markers through whole-cell proteomics confirmed their comparable enrichment in the three lines, demonstrating that the new method can be applied to iPSC lines from different individuals to standardize DA neuron cultures. While we focus on methodological developments here, a companion analysis of the same whole-cell proteomics dataset, examining molecular features potentially associated with disease statuses, has been reported separately.
SN - 1422-0067
ER -