In Vitro Induction of Neuromuscular Junctions from Engineered Human-Induced Pluripotent Stem Cells

Abstract

Source:Lin, C. et al., In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells. J. Vis. Exp.(2020).

This experiment details a method for generating neuromuscular junctions from human-induced pluripotent stem cells. It uses a staged differentiation process to promote myotube and motor neuron development and synapse formation, yielding functional neuromuscular junctions.

Protocol

1. Preparation of extracellular matrix (ECM)-coated plates

1. Dilute ECM (see Table of Materials) with ice cold 1x PBS to a final concentration of 2%
2. Add 1 mL of ECM to 49 mL of 1x PBS in a 50 mL plastic tube. Mix well by pipetting up and down several times.
3. Place 1 coverslip into each well of a 6-well plate. Add 1.5 mL of 2% ECM into each well.
4. Incubate the well plate with 2% ECM for 2 h at 37 °C.
5. Aspirate ECM from the well plate and store the plate at 4 °C before use.

2. Differentiation of iPSCs toward NMJ

1. Seed ....

Results

Table 1: Formula of medium.

Myogenic differentiation medium (MDM)	MEM-alpha	500mL
	100mM 2-ME	1117μL
	Doxycycline	1μg/mL
	KSR	56mL (final to 10%)
	P.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medium, growth factors and reagents
2-mercaptoethanol	Nacalai tesque	21418-42
B27, Gibco	Gibco	12587-001
BDNF	R & D Systems	248-BD
Cell detachment solution, Accumax	STEMCELL Technologies	#07921
Critical point dryer	Hitachi	ES-2030
Doxycycline	Takara	631311
ECM, Matrigel (growth factor reduced)	Corning	356230
GDNF	R & D Systems	212-GD
Gelation, IP gel	Geno Staff	PG20-1
Ions coater	JEOL	JEC3000FC
iPSC medium, mTeSR	STEMCELL Technologies	85850
KnockOut SR (KSR)	Gibco	10828-028
live cell microscopy	Nikon		Eclipse Ti microscope
MEM-alpha	Gibco	12571-071
N2, Gibco	Gibco	17502-048
Neurobasal medium	Gibco	21103-049
NT3	R & D Systems	267-N3
Primate ES cell medium	ReproCell	RCHEMD001
SEM	Hitachi	S-4700
TEM	Hitachi	H7650
Y27632	Wako Chemicals GmbH	253-00513