Differentiation protocol and maturation shape the axial identity and synaptic state in human iPSC-derived spinal motor neurons
- 1Cedars-Sinai Board of Governors Regenerative Medicine Institute
Abstract
Human induced pluripotent stem cell (iPSC)-derived motor neurons are widely used for disease modeling. Stem cells can be differentiated into motor neurons by exposure to specific patterning morphogens, which can ultimately determine terminal cellular composition and developmental state. We compared two small molecule workflows for human iPSC maturation into motor neurons: a rapid direct protocol (diMN protocol) and an extended protocol (purMN protocol), with both protocols matched for 20 days in maturation medium. While the diMN protocol generated mixed neural cultures, the purMN protocol provided cultures with greater motor neuron enrichment, stronger caudal spinal identity, broader synaptic and cholinergic programs, and reduced progenitor-associated signatures. By extending the purMN protocol to 32 days in maturation media, cultures developed reinforced spinal, postsynaptic, presynaptic, calcium-signaling, and cholinergic features. Across four independent reference frameworks, deconvolution aligned diMN cultures with anterior and progenitor-associated states and purMN cultures with spinal and post-mitotic states. In a microfluidic co-culture system, purMNs formed denser, more highly branched distal neurite networks and produced more innervated acetylcholine receptor clusters compared to diMNs. Revealing how purity and maturation jointly define the transcriptomic and structural state of human iPSC-derived spinal motor neuron cultures will help the field choose optimal models for studies of neurodevelopment and neurodegenerative diseases.
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BibTeX
@misc{sansa2026differentiation,
title = {{Differentiation protocol and maturation shape the axial identity and synaptic state in human iPSC-derived spinal motor neurons}},
author = {A. Sansa and V. A. Zhemkov and M. Feole and J. M. Villalba and S. Bell and C. N. Svendsen},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751550},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751550v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Differentiation protocol and maturation shape the axial identity and synaptic state in human iPSC-derived spinal motor neurons
AU - A. Sansa
AU - V. A. Zhemkov
AU - M. Feole
AU - J. M. Villalba
AU - S. Bell
AU - C. N. Svendsen
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751550
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751550v1
AB - Human induced pluripotent stem cell (iPSC)-derived motor neurons are widely used for disease modeling. Stem cells can be differentiated into motor neurons by exposure to specific patterning morphogens, which can ultimately determine terminal cellular composition and developmental state. We compared two small molecule workflows for human iPSC maturation into motor neurons: a rapid direct protocol (diMN protocol) and an extended protocol (purMN protocol), with both protocols matched for 20 days in maturation medium. While the diMN protocol generated mixed neural cultures, the purMN protocol provided cultures with greater motor neuron enrichment, stronger caudal spinal identity, broader synaptic and cholinergic programs, and reduced progenitor-associated signatures. By extending the purMN protocol to 32 days in maturation media, cultures developed reinforced spinal, postsynaptic, presynaptic, calcium-signaling, and cholinergic features. Across four independent reference frameworks, deconvolution aligned diMN cultures with anterior and progenitor-associated states and purMN cultures with spinal and post-mitotic states. In a microfluidic co-culture system, purMNs formed denser, more highly branched distal neurite networks and produced more innervated acetylcholine receptor clusters compared to diMNs. Revealing how purity and maturation jointly define the transcriptomic and structural state of human iPSC-derived spinal motor neuron cultures will help the field choose optimal models for studies of neurodevelopment and neurodegenerative diseases.
ER -