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BRAIN Publication-derived

Cortico-Motor Assembloid with Functional Neuromuscular

Source Andersen et al. · Stanford University School of Medicine, Stanford, CA, USA · 10.1016/j.cell.2020.11.017

👤 Andersen J, Revah O, Miura Y, Thom N, Amin ND, Kelley KW, Singh M, Chen X, Thete MV, Walczer EM, Bhatt SM, Bhatt AH, Paşca SP ⏱ 70 days 📋 8 phases 🧫 Human iPSC

Abstract

Andersen et al. created a three-component assembloid system combining cortical, spinal, and skeletal muscle spheroids to model the complete voluntary motor pathway. Cortical stimulation evoked muscle contractions through spinal motor neuron relay, demonstrating functional corticospinal-neuromuscular connectivity in human tissue for the first time.

Cell source
Human iPSC
Application
Corticospinal Tract Development

Protocol overview

35 steps across 8 phases

Generation of human cortical spheroids (hCS) Days 0-42+
  1. 1 Culture hiPS cells on feeder layers
  2. 2 Initiate spheroid formation
  3. 3 Transition to neural medium
  4. 4 Add growth factors for maturation
  5. 5 Maintain mature hCS
Generation of human hindbrain/cervical spinal cord spheroids (hSpS) Days 0-50+
  1. 1 Initiate neural induction with dual SMAD inhibition
  2. 2 Add WNT activator and establish rostro-caudal patterning
  3. 3 Add SHH modulator for ventral specification
  4. 4 Switch to maturation medium and apply Notch inhibitor
  5. 5 Maintain long-term hSpS culture
Generation of 3D human skeletal muscle (hSkM) Days 0-25
  1. 1 Culture human skeletal myoblasts in growth medium
  2. 2 Dissociate myoblasts for 3D culture
  3. 3 Seed 3D hSkM in silicone wells and initiate gelation
  4. 4 Transfer to ultra-low attachment plates for differentiation
  5. 5 Switch to differentiation medium
Assembly of cortico-spinal assembloids (hCS-hSpS) Days 0-30+
  1. 1 Prepare hCS and hSpS for assembly
  2. 2 Fuse spheroids in close proximity
  3. 3 Change medium on day 2 of assembly
  4. 4 Transfer to culture plates on day 3
  5. 5 Maintain long-term culture
Generation of spinal-muscle assembloids (hSpS-hSkM) Days 0-20+
  1. 1 Prepare 3D hSkM for assembly
  2. 2 Prepare transwell insert setup
  3. 3 Place hSpS on insert in contact with hSkM
  4. 4 Change medium and maintain culture
Assembly of three-part cortico-motor assembloids (hCS-hSpS-hSkM) Days 0-70+
  1. 1 Pre-assemble hSpS-hSkM
  2. 2 Add hCS to established hSpS-hSkM assembloid
  3. 3 Maintain three-part assembloid
Viral labeling and transduction Days -7 to +0 (relative to assembly)
  1. 1 Prepare spheroid for viral infection
  2. 2 Add fresh medium and transfer to culture plate
  3. 3 Assess viral expression before assembly
Functional analysis—Imaging and electrophysiology Days 5-70 post-assembly (variable based on analysis type)
  1. 1 Live imaging of eYFP projections in intact assembloids
  2. 2 Optogenetic stimulation with GCaMP imaging
  3. 3 Patch-clamp electrophysiology in sliced assembloids
  4. 4 Glutamate uncaging on hSkM
  5. 5 Optogenetic stimulation of hCS with muscle contraction readout

Full SOP

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Attribution

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

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