2. Academic Validation
  3. In vitro derivation of midbrain dopaminergic neurons from porcine embryonic stem cells in multi-dimensional conditions

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

  • Stem Cell Res Ther. 2025 Nov 5;16(1):617. doi: 10.1186/s13287-025-04693-9.
Hyerin Choi 1 2 3,  Dongjin Oh 1 2 3,  Ali Jawad 1 2,  Zheng Haomiao 1 2,  Jaehyung Ham 1 2,  Juyoung Heo 1 2,  Aram Oh 1 2,  Huijin Jin 1 2,  Jaehyeok Seo 1 2,  Byoung Chol Oh 4,  Sang-Hwan Hyun 5 6 7 8 9
Affiliations

Affiliations

  • 1 Veterinary Medical Center, College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju, Republic of Korea.
  • 2 Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea.
  • 3 Department of Neurology, Institute for Cell Engineering, School of Medicine, Johns Hopkins Medicine, Baltimore, ML, USA.
  • 4 Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • 5 Veterinary Medical Center, College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju, Republic of Korea. [email protected].
  • 6 Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea. [email protected].
  • 7 Vet-ICT Convergence Education and Research Center (VICERC), Chungbuk National University, Cheongju, Republic of Korea. [email protected].
  • 8 Chungbuk National University Hospital, Cheongju, Republic of Korea. [email protected].
  • 9 Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, 28644, Republic of Korea. [email protected].
PMID: 41194288 DOI: 10.1186/s13287-025-04693-9
Abstract

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.

Keywords

Dopaminergic neuron; Embryonic stem cells; Midbrain; Organoid; Porcine.

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