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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids for Regenerative Medicine

Full SOP with Academic or a plan

Source Kim et al., 2021 · Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Republic of Korea · 10.1016/j.pneurobio.2021.102086

Authors: Seung Won Kim, Hye-Ji Woo, Eun Hee Kim, Hyung Sun Kim, Han Na Suh, Soo-hyun Kim, Jae-Jin Song, Noviana Wulansari, Minji Kang, Se-Young Choi, Su Jeong Choi, Won Hyuk Jang, Jungbin Lee, Ki Hean Kim, Wongyoung Lee, Sung Hyun Kim, Jinhee Yang, Jangbeen Kyung, Hyun-Seob Lee, Sang Myun Park, Mi-Yoon Chang, Sang-Hun Lee 7 phases Cell source: Human hESC and hiPSC

Abstract

This protocol describes the derivation of ventral midbrain-type neural stem/precursor cells (Og-NSCs) from human pluripotent stem cell-derived midbrain organoids in 3D culture. These NSCs can be stably expanded and differentiated into midbrain-type dopamine neurons with high expression of midbrain-specific markers (FOXA2, LMX1A, NURR1, EN1) and low cell-to-cell variability, providing a scalable, reproducible source for Parkinson's disease modeling and cell therapy.

Cell source
Human hESC and hiPSC
Application
Disease modeling and cell therapy for Parkinson's disease

Protocol overview

18 steps across 7 phases

Phase 1 · hPSC culture and maintenance Ongoing
  1. a Plate hESCs/iPSCs on vitronectin-coated dishes
  2. b Maintain undifferentiated hPSC cultures
Phase 2 · Midbrain-like organoid formation in 3D (Days 1-10) Day 1-10
  1. a Seed hESCs in low-attachment plates for neural induction
  2. b Prepare neural induction medium (N2)
  3. c Initiate ventral patterning (Days 1-10)
Phase 3 · Midbrain organoid expansion and NSC derivation (Days 10-18) Day 10-18
  1. a Transfer spheres to 6-well plates
  2. b Prepare modified organoid protocol for VM-NSC enrichment
Phase 4 · Organoid-derived NSC (Og-NSC) isolation and expansion (Day 18 onward) Day 18–ongoing
  1. a Dissociate and plate organoids on vitronectin
  2. b Expand Og-NSCs with passage intervals
  3. c Store Og-NSCs in liquid nitrogen
Phase 5 · Terminal differentiation of Og-NSCs into midbrain dopamine neurons Day 0-15 (differentiation)
  1. a Induce terminal differentiation of Og-NSCs
  2. b Monitor neuronal differentiation and maturation
Phase 6 · Immunostaining for marker analysis Variable (typically post-differentiation)
  1. a Fix cells for immunocytochemistry
  2. b Incubate with primary antibodies
  3. c Incubate with secondary antibodies and mount
  4. d Image stained cells
Phase 7 · Optional: Organoid cryosectioning and immunostaining Variable (post-organoid formation)
  1. a Fix and prepare organoids for sectioning
  2. b Prepare cryosections and immunostain

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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