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OTHER Publication-derived

Spinal Motor Organoid Differentiation from MELAS Patient-Derived iPSCs with Notch Inhibition

Source Winanto et al., 2020 · Institute of Molecular and Cell Biology, A*STAR Research Entities, Singapore · 10.1038/s41419-020-2383-6

👤 Winanto, Zi Jian Khong, Boon-Seng Soh, Yong Fan, Shi-Yan Ng ⏱ 42 days 📋 10 phases 🧫 Patient-Derived iPSC (MELAS with A3243G mtDNA mutation)

Abstract

This protocol generates spinal motor neuron organoids from patient-derived iPSCs carrying the MELAS-associated A3243G mitochondrial DNA mutation. The method reveals hyperactive Notch signaling underlying motor neuron differentiation defects and demonstrates that gamma-secretase inhibition with DAPT reverses neurodevelopmental and neurite outgrowth impairments in the disease model.

Cell source
Patient-Derived iPSC (MELAS with A3243G mtDNA mutation)
Application
Disease modeling and neurodevelopmental defect investigation

Protocol overview

45 steps across 10 phases

iPSC Maintenance and Pluripotency Verification Ongoing
  1. 1 Maintain iPSCs on Matrigel-coated plates in feeder-free culture
  2. 2 Verify iPSC pluripotency by immunostaining
Neural Progenitor Cell (NPC) Induction Days 0–10
  1. 1 Dissociate iPSC colonies and seed for neural induction
  2. 2 Apply BMP inhibition and Wnt activation (Days 0–10)
  3. 3 Introduce retinoic acid for caudalization (Days 3–10)
  4. 4 Verify NPC differentiation at Day 10
Embryoid Body Formation and Organoid Maturation in Suspension Days 10–28
  1. 1 Encapsulate neuralized cells in Matrigel droplets (Day 10)
  2. 2 Transfer organoids to spinner flask for maturation (Day 14)
  3. 3 Maintain organoid culture in spinner flask maturation media (Days 14–28)
Motor Neuron Progenitor Specification (Alternative 2D Protocol) Days 11–28 (in parallel 2D culture)
  1. 1 Continue differentiation with RA and Purmorphamine (Days 11–17)
  2. 2 Supplement with BDNF and GDNF for motor neuron maturation (Days 17–28)
  3. 3 Verify motor neuron generation at Day 28 (2D protocol endpoint)
Rotenone Complex I Inhibition Studies (Days 18–28) Days 18–28
  1. 1 Harvest organoids and transfer to six-well plates on Day 18
  2. 2 Treat organoids with Rotenone (0.25 μM, Days 18–28)
  3. 3 Maintain organoids on orbital shaker during treatment
  4. 4 Harvest organoids at Day 28 for gene expression analysis
DAPT Notch Inhibition Treatment (Days 18–28) Days 18–28
  1. 1 Harvest organoids and transfer to six-well plates on Day 18
  2. 2 Treat organoids with DAPT (2.5 μM, Days 18–28)
  3. 3 Maintain organoids on orbital shaker during DAPT treatment
  4. 4 Assess Notch inhibition at Day 28 by qPCR
  5. 5 Fix organoids at Days 28 and 35 for immunostaining analysis
Organoid Immunostaining and Morphological Analysis Days 21–42 (at harvest timepoints)
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Serially dehydrate organoids in sucrose solutions
  3. 3 Embed organoids in OCT compound and cryosection
  4. 4 Permeabilize cryosections
  5. 5 Block non-specific antibody binding
  6. 6 Incubate with primary antibodies overnight at 4°C
  7. 7 Incubate with secondary antibodies
  8. 8 Stain nuclei with DAPI
  9. 9 Mount sections and acquire fluorescence images
Neurite Outgrowth Analysis Days 21–28
  1. 1 Seed organoids onto Matrigel-coated plates at Day 21
  2. 2 Culture organoids for neurite outgrowth for 7 days (Days 21–28)
  3. 3 Fix organoids with paraformaldehyde at Day 28
  4. 4 Immunostain for SMI-32 motor neuron axon marker
  5. 5 Measure neurite lengths using image analysis
RNA Extraction and Gene Expression Analysis by qPCR Days 21–42
  1. 1 Pool organoids for RNA extraction
  2. 2 Extract RNA using Trizol reagent
  3. 3 Purify RNA using RNeasy Mini Kit
  4. 4 Reverse transcribe RNA to cDNA
  5. 5 Design and validate qPCR primer sets
  6. 6 Perform qPCR on QuantStudio 5 Real-Time PCR System
  7. 7 Normalize gene expression and analyze results
Heteroplasmy Assessment by RFLP Throughout culture
  1. 1 Perform initial deep sequencing of mtDNA
  2. 2 Assess heteroplasmy levels during differentiation (Day 10 NPCs)
  3. 3 Monitor heteroplasmy levels during passage

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Winanto et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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