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BRAIN Publication-derived

Familial Parkinson's Disease Midbrain Organoids

Full SOP with Academic or a plan

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

Authors: Andrea Becerra-Calixto, Abhisek Mukherjee, Santiago Ramirez, Sofia Sepulveda, Tirthankar Sinha, Rabab Al-Lahham, Nicole De Gregorio, Camila Gherardelli, Claudio Soto Duration: 180 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Becerra-Calixto et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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