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BRAIN Publication-derived

Generation of Human Midbrain-Like Organoids (hMBOs) from hiPSCs for Parkinson's Disease Modeling

Source Becerra-Calixto et al., 2023 · Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston · 10.3390/cells12040625

👤 Andrea Becerra-Calixto, Abhisek Mukherjee, Santiago Ramirez, Sofia Sepulveda, Tirthankar Sinha, Rabab Al-Lahham, Nicole De Gregorio, Camila Gherardelli, Claudio Soto ⏱ 180 days 📋 7 phases 🧫 Patient-Derived iPSC (Familial Parkinson's Disease with SNCA triplication)

Abstract

This protocol describes the generation of human midbrain-like organoids (hMBOs) from induced pluripotent stem cells (hiPSCs) derived from a familial Parkinson's disease patient carrying SNCA gene triplication. The resulting organoids spontaneously accumulate pathological α-synuclein into Lewy body-like inclusions and exhibit loss of dopaminergic neurons, providing a three-dimensional in vitro model to study Parkinson's disease pathogenesis.

Cell source
Patient-Derived iPSC (Familial Parkinson's Disease with SNCA triplication)
Application
Disease modeling

Protocol overview

43 steps across 7 phases

hiPSC Culture Maintenance Ongoing
  1. 1 Coat plates with hESC-Qualified Matrigel
  2. 2 Culture hiPSCs in mTeSR Plus medium
  3. 3 Perform three passages before organoid generation
  4. 4 Perform monthly mycoplasma testing
Embryoid Body Formation and Neural Induction Day 0–2
  1. 1 Dissociate hiPSC colonies into single-cell suspension
  2. 2 Form embryoid bodies in 96-well ultra-low attachment plates
  3. 3 Culture EBs in neural induction medium
  4. 4 Replace medium on day 2
Midbrain Patterning Day 4–7
  1. 1 Supplement EBs with midbrain patterning factors on day 4
  2. 2 Culture EBs with midbrain factors for 3 days
  3. 3 Embed EBs in reduced growth factor Matrigel on day 7
Neuroectodermal Spheroid Maturation Day 7–9
  1. 1 Culture embedded spheroids in tissue growth induction medium
  2. 2 Supplement with additional factors on day 7
  3. 3 Transfer organoids to ultra-low attachment 6-well plates on day 9
hMBO Differentiation and Maturation Day 9–180
  1. 1 Culture hMBOs in organoid differentiation medium
  2. 2 Place plates on orbital shaker at 70 rpm
  3. 3 Replace medium every 3–4 days
  4. 4 Culture hMBOs for up to 180 days for pathology analysis
Fixation and Immunohistochemistry (IHC) Upon harvest
  1. 1 Fix hMBOs in 4% paraformaldehyde
  2. 2 Dehydrate samples using graded ethanol and xylenes
  3. 3 Embed samples in paraffin
  4. 4 Cut paraffin sections using a microtome
  5. 5 Rehydrate slides
  6. 6 Perform antigen retrieval
  7. 7 Block endogenous peroxidase activity
  8. 8 Block non-specific binding
  9. 9 Incubate with primary antibodies overnight
  10. 10 Wash slides with PBS
  11. 11 Incubate with HRP-linked secondary antibodies
  12. 12 Wash slides with PBS
  13. 13 Visualize peroxidase reaction with DAB chromogen
  14. 14 Counterstain with hematoxylin
  15. 15 Dehydrate and mount slides
  16. 16 Acquire images using confocal microscope
Immunofluorescence (IF) Staining Upon harvest
  1. 1 Deparaffinize and hydrate paraffin sections
  2. 2 Block non-specific binding with donkey serum
  3. 3 Incubate with primary antibodies overnight
  4. 4 Wash slides with PBS
  5. 5 Incubate with fluorescent secondary antibodies
  6. 6 Wash slides with PBS and apply DAPI
  7. 7 Mount with FluorSave and apply coverslip
  8. 8 Acquire confocal and epifluorescence images
  9. 9 Quantify immunostaining

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Becerra-Calixto et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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