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MUSCLE Publication-derived

Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons

Source Osaki et al., 2018 · Massachusetts Institute of Technology, Department of Mechanical Engineering · 10.1126/sciadv.aat5847

👤 Tatsuya Osaki, Sebastien G. M. Uzel, Roger D. Kamm ⏱ 42 days 📋 10 phases 🧫 Patient-Derived iPSC (Sporadic ALS with G298S TDP-43 mutation)

Abstract

This protocol describes the development of a 3D motor unit model on a microfluidic chip using patient-derived ALS iPSC motor neurons and human skeletal muscle bundles. The model recapitulates key ALS pathological features including reduced muscle contraction, motor neuron degradation, and increased muscle apoptosis, and demonstrates drug screening capability with optogenetic stimulation for measuring contractile responses.

Cell source
Patient-Derived iPSC (Sporadic ALS with G298S TDP-43 mutation)
Application
Disease modeling and drug screening for ALS

Protocol overview

51 steps across 10 phases

NSC Maintenance and Motor Neuron Differentiation Day 0 to Day 42
  1. 1 Maintain hESC and ALS-iPSC-derived NSCs on basement membrane
  2. 2 Form neurospheres in 96-well spindle-bottom plates
  3. 3 Initiate MN differentiation with caudalization and ventralization factors
  4. 4 Continue MN differentiation without activin A
  5. 5 Mature motor neurons with BDNF and GDNF
  6. 6 Remove neural progenitor cells from spheroids
Transfection of Channelrhodopsin-2 into NSCs Day 27-28 (concurrent with final maturation)
  1. 1 Plate hESC and ALS-iPSC NSCs for transfection
  2. 2 Incubate cells with AAV particles containing ChR2 plasmid
  3. 3 Culture transfected cells for expansion
  4. 4 Sort transfected cells by FACS
  5. 5 Replate sorted ChR2-NSCs and form neurospheres
Skeletal Muscle Differentiation in Microfluidic Device Day 0 to Day 13
  1. 1 Prepare microfluidic device and prevent cell attachment to glass
  2. 2 Inject iPSC-derived skeletal myoblasts with collagen/Matrigel
  3. 3 Allow muscle fiber bundle formation around pillar structures
  4. 4 Apply skeletal myocyte differentiation medium
  5. 5 Continue differentiation with medium changes
Motor Neuron Spheroid Injection and Coculture Initiation Day 13 to Day 14 (injection day)
  1. 1 Prepare predifferentiated MN spheroids for injection
  2. 2 Inject MN spheroids with collagen gel into left compartment
  3. 3 Establish segregated culture media in separate reservoirs
Motor Unit Maturation and Neuromuscular Junction Formation Day 14 to Day 21 (coculture phase)
  1. 1 Observe motor neurite outgrowth initiation
  2. 2 Monitor neurite reaching muscle fiber bundle
  3. 3 Verify NMJ formation by immunostaining nAChR clusters
  4. 4 Confirm functional NMJ by chemical stimulation assay
  5. 5 Measure spontaneous muscle contraction frequency and force
Glutamate Excitotoxicity Treatment (to model ALS pathogenesis) Day 14-21 of coculture
  1. 1 Add high-concentration glutamic acid for excitotoxicity induction
  2. 2 Assess morphological changes in motor neurons
  3. 3 Measure reduced muscle contraction force under glutamate treatment
  4. 4 Rinse and recover from brief TTX treatment (optional neurotoxin test)
ALS Patient Motor Unit Establishment Day 14-28 of coculture (using ALS-iPSC MNs)
  1. 1 Characterize ALS patient-derived MN spheroids before injection
  2. 2 Inject ALS-MN spheroids into microfluidic devices with muscle
  3. 3 Monitor ALS motor unit maturation and NMJ formation
  4. 4 Measure baseline ALS motor unit contractility
  5. 5 Assess muscle apoptosis in ALS motor units
Drug Treatment and Efficacy Assessment (Rapamycin and Bosutinib) Day 4-14 of ALS motor unit coculture
  1. 1 Initiate drug treatment on day 4 of coculture
  2. 2 Measure drug effects on ALS motor unit muscle contraction at day 7
  3. 3 Measure significant drug neuroprotection at day 14
  4. 4 Quantify optical stimulation success rate (muscle contraction reliability)
  5. 5 Assess muscle apoptosis reduction with drug treatment
Analysis of Autophagy Pathway Activation and TDP-43 Degradation Day 14 of ALS motor unit coculture
  1. 1 Isolate tissues and extract RNA for qRT-PCR
  2. 2 Perform reverse transcription and qRT-PCR for autophagy genes
  3. 3 Measure TDP-43 mRNA expression reduction
  4. 4 Immunostain motor neurons for TDP-43 aggregation status
  5. 5 Analyze myogenic and apoptotic gene expression in muscle
Drug Treatment via Endothelial Cell Barrier (Blood-Brain Barrier Model) Day 0-7 (EC differentiation), Day 7-14 (drug treatment with EC barrier)
  1. 1 Prepare microfluidic device and inject type I collagen in left compartment
  2. 2 Seed iPSC-derived endothelial cells on collagen layer
  3. 3 Differentiate ECs toward brain-specific phenotype with retinoic acid
  4. 4 Verify EC barrier formation by tight junction staining
  5. 5 Confirm low permeability of EC barrier to large molecules
  6. 6 Apply drugs (rapamycin and bosutinib) to left side of EC barrier
  7. 7 Measure muscle contraction force with EC barrier present
  8. 8 Assess P-glycoprotein expression changes with drug treatment

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Osaki et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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