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Background <p>. Porcine pluripotent stem cells hold significant promise as a large-animal model for human neurodevelopment and disease modeling. However, efficient protocols for their directed differentiation into midbrain dopaminergic (mDA) neurons and the generation of 3D midbrain-like organoids remain limited. This study aimed to establish species-optimized conditions for the derivation of functional mDA neurons from porcine embryonic stem cells (pESCs) under both two- and three-dimensional environments.</p> Methods <p>. In vitro fertilization (IVF)- and parthenogenetic activation (PA)-derived pESCs were subjected to neural induction using stepwise exposure to dual SMAD inhibition, SHH, CHIR99021, and FGF8. For monolayer culture, adherent monolayers were differentiated on Matrigel- or poly-L-ornithine/laminin I/fibronectin-coated surfaces. For 3D culture, porcine midbrain-like organoids (pMLOs) were formed under low-adhesion conditions. Functional and molecular characterization was performed via immunofluorescence, patch-clamp electrophysiology, microelectrode array (MEA) recordings, dopamine ELISA, and RNA sequencing.</p> Results <p>. Porcine embryonic stem cells required higher SHH and GSK3 inhibition thresholds to efficiently induce FOXA2⁺ ventral midbrain progenitors, revealing a species-specific divergence from human protocols. IVF-derived pESCs showed markedly enhanced dopaminergic differentiation and functional maturation compared to PA-derived pESCs. Notably, in 3D culture, neuroepithelial structures emerged as early as day 5, and functionally mature mDA neurons—confirmed by TH expression, dopamine release, and spontaneous synaptic activity—were detected by day 28. This timeline represents an accelerated maturation relative to comparable human mDA differentiation systems, where functional properties typically arise after day 35. Single-cell RNA sequencing further delineated dynamic dopaminergic lineage trajectories and revealed porcine-specific gene expression patterns associated with early mDA identity acquisition.</p> Conclusions <p>. This study presents the first robust platform for generating functionally validated mDA neurons and midbrain-like organoids from porcine stem cells. The findings highlight species-specific signaling dynamics and establish porcine in vitro models as scalable, translational systems for investigating neurodevelopment and disease mechanisms.</p>

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In vitro derivation of midbrain dopaminergic neurons from porcine embryonic stem cells in multi-dimensional conditions

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月5日

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  • Hyerin Choi,
  • Dongjin Oh,
  • Ali Jawad,
  • Zheng Haomiao,
  • Jaehyung Ham,
  • Juyoung Heo,
  • Aram Oh,
  • Huijin Jin,
  • Jaehyeok Seo,
  • Byoung Chol Oh,
  • Sang-Hwan Hyun

摘要

Background

. Porcine pluripotent stem cells hold significant promise as a large-animal model for human neurodevelopment and disease modeling. However, efficient protocols for their directed differentiation into midbrain dopaminergic (mDA) neurons and the generation of 3D midbrain-like organoids remain limited. This study aimed to establish species-optimized conditions for the derivation of functional mDA neurons from porcine embryonic stem cells (pESCs) under both two- and three-dimensional environments.

Methods

. In vitro fertilization (IVF)- and parthenogenetic activation (PA)-derived pESCs were subjected to neural induction using stepwise exposure to dual SMAD inhibition, SHH, CHIR99021, and FGF8. For monolayer culture, adherent monolayers were differentiated on Matrigel- or poly-L-ornithine/laminin I/fibronectin-coated surfaces. For 3D culture, porcine midbrain-like organoids (pMLOs) were formed under low-adhesion conditions. Functional and molecular characterization was performed via immunofluorescence, patch-clamp electrophysiology, microelectrode array (MEA) recordings, dopamine ELISA, and RNA sequencing.

Results

. Porcine embryonic stem cells required higher SHH and GSK3 inhibition thresholds to efficiently induce FOXA2⁺ ventral midbrain progenitors, revealing a species-specific divergence from human protocols. IVF-derived pESCs showed markedly enhanced dopaminergic differentiation and functional maturation compared to PA-derived pESCs. Notably, in 3D culture, neuroepithelial structures emerged as early as day 5, and functionally mature mDA neurons—confirmed by TH expression, dopamine release, and spontaneous synaptic activity—were detected by day 28. This timeline represents an accelerated maturation relative to comparable human mDA differentiation systems, where functional properties typically arise after day 35. Single-cell RNA sequencing further delineated dynamic dopaminergic lineage trajectories and revealed porcine-specific gene expression patterns associated with early mDA identity acquisition.

Conclusions

. This study presents the first robust platform for generating functionally validated mDA neurons and midbrain-like organoids from porcine stem cells. The findings highlight species-specific signaling dynamics and establish porcine in vitro models as scalable, translational systems for investigating neurodevelopment and disease mechanisms.