Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons
- 1Memorial Sloan Kettering Cancer Center
Abstract
Midbrain dopaminergic (mDA) neurons comprise molecularly and functionally distinct subtypes with differential vulnerability in neurodegenerative and psychiatric disorders. However, the mechanisms specifying subtype identity remain poorly understood, and protocols for the selective derivation of human mDA subtypes are lacking. Here we establish a strategy to derive substantia nigra (A9) and ventral tegmental area (A10) mDA neurons from human pluripotent stem cells (hPSCs). A9 identity is specified by dual-SMAD activation through Activin A and BMP7 at the midbrain floor-plate progenitor stage, whereas A10 identity is promoted by BMP inhibition. A9 mDA neurons are purified based on ALDH1A1 expression, and subtype identity is maintained by continued TGF-{beta} modulation and ESRRB activation in vitro and upon transplantation in vivo. Single-cell RNA sequencing and biochemical analyses demonstrate that hPSC-derived A9 neurons exhibit increased oxidative phosphorylation, neuromelanin-like pigmentation, elevated dopamine synthesis and release, and electrophysiological properties characteristic of A9 mDA neurons in vivo. Integration with human fetal midbrain datasets confirms strong transcriptional concordance between in vitro-derived and in vivo mDA subtypes. Neuromelanin-like structures produced by hPSC-derived A9 neurons trigger pro-inflammatory cytokine secretion from hPSC-derived microglia, and A9 neurons are primed to upregulate MHC-I genes in response to interferon-{gamma}, features which may contribute to the selective vulnerability of A9 neurons. Together, these results establish a robust in vitro platform to interrogate human mDA subtype development, function, and selective vulnerability, enabling mechanistic studies relevant to Parkinson's disease and the development of cell-based therapies.
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BibTeX
@misc{yang2026decoding,
title = {{Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons}},
author = {D. Yang and S. Choi and J. Piao and A. Evangelisti and Z. Yang and V. D. Bocchi and S. A. Donohue and N. Claros and J. Jungverdorben and M. Sidharta and Y. Wu and H. A. Pasolli and T. W. Kim and T. Zhou and D. Sulzer and E. Mosharov and V. Tabar and L. Studer},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753784},
url = {https://www.biorxiv.org/content/10.64898/2026.09.23.753784v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons
AU - D. Yang
AU - S. Choi
AU - J. Piao
AU - A. Evangelisti
AU - Z. Yang
AU - V. D. Bocchi
AU - S. A. Donohue
AU - N. Claros
AU - J. Jungverdorben
AU - M. Sidharta
AU - Y. Wu
AU - H. A. Pasolli
AU - T. W. Kim
AU - T. Zhou
AU - D. Sulzer
AU - E. Mosharov
AU - V. Tabar
AU - L. Studer
PY - 2026
DA - 2026/09/23
PB - bioRxiv
DO - 10.64898/2026.09.23.753784
UR - https://www.biorxiv.org/content/10.64898/2026.09.23.753784v1
AB - Midbrain dopaminergic (mDA) neurons comprise molecularly and functionally distinct subtypes with differential vulnerability in neurodegenerative and psychiatric disorders. However, the mechanisms specifying subtype identity remain poorly understood, and protocols for the selective derivation of human mDA subtypes are lacking. Here we establish a strategy to derive substantia nigra (A9) and ventral tegmental area (A10) mDA neurons from human pluripotent stem cells (hPSCs). A9 identity is specified by dual-SMAD activation through Activin A and BMP7 at the midbrain floor-plate progenitor stage, whereas A10 identity is promoted by BMP inhibition. A9 mDA neurons are purified based on ALDH1A1 expression, and subtype identity is maintained by continued TGF-{beta} modulation and ESRRB activation in vitro and upon transplantation in vivo. Single-cell RNA sequencing and biochemical analyses demonstrate that hPSC-derived A9 neurons exhibit increased oxidative phosphorylation, neuromelanin-like pigmentation, elevated dopamine synthesis and release, and electrophysiological properties characteristic of A9 mDA neurons in vivo. Integration with human fetal midbrain datasets confirms strong transcriptional concordance between in vitro-derived and in vivo mDA subtypes. Neuromelanin-like structures produced by hPSC-derived A9 neurons trigger pro-inflammatory cytokine secretion from hPSC-derived microglia, and A9 neurons are primed to upregulate MHC-I genes in response to interferon-{gamma}, features which may contribute to the selective vulnerability of A9 neurons. Together, these results establish a robust in vitro platform to interrogate human mDA subtype development, function, and selective vulnerability, enabling mechanistic studies relevant to Parkinson's disease and the development of cell-based therapies.
ER -