An In Vitro Method to Generate Human Brain Organoids

Samenvatting

Source: Gabriel, E., et al., Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders. J. Vis. Exp. (2017)

The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Generation of Brain Organoids (23 days)

1. Initiation of neuroectoderm (5 days)
   NOTE: The following points should be considered before the start of differentiation. The reprogramming method (lentiviral-, sendai-virus-, episomal-, or microRNA-based etc.) to obtain human iPSCs should ideally be the same for all patient and control iPSC lines. Various reprogramming kits and instructions based on published protocols are available. The quality of the human....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DMEM/F-12	Gibco, US	31331093	Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
Dorsomorphin	Sigma-Aldrich, US	P5499	Compound C
Embedding medium	AppliChem	A9011, 0100	Mowiol; embedding medium; multiple suppliers
Engelbreth-Holm-Swarm (EHS) matrix	Corning	354277	Matrigel hESC-qualified matrix; important: hESC qualified
Insulin	Sigma-Aldrich, US	I3536-100MG
L-glutamine	Gibco, US	25030081	L-glutamine (200 mM)
Medium A	Stem cell technologies	#05850	mTeSR1 (hiPSC medium)
Medium B	Stem cell technologies	#05835	Neural induction medium (NIM); neural differentiation medium
Medium C	Gibco, US	21103049	Neural Basal Medium
MEM	Gibco, US	11140035	MEM non-essential amino acids solution (100x)
MycoAlert Mycoplasma Detection Kit	Lonza, Switzerland	#LT07-218	Mycoplasma detection kit; multiple suppliers
Penicillin-Streptomycin (10.000 U/ml)	Gibco, US	15140122
Reagent A	Stem cell technologies	# 05872	ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer´s protocol
Reagent B	Sigma-Aldrich, US	A6964-100ML	Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 "enzymatic cell dissociation solution"
Supplement 1	Gibco, US	17502048	N-2 supplement (100x)
Supplement 2 w/o Vitamin A	Gibco, US	12587010	B-27 supplement (50x), minus vitamin A
SB431542	Selleckchem.com	S1067
Spinner flask 250 ml	IBS Integra Bioscience	182026
waterproof sheet	BEMIS company, inc.	PM996	Parafilm "M"
Y-27632	Selleckchem.com	S1049	ROCK-inhibitor (Y-27632 2HCL)
β-mercaptoethanol	Gibco, US	31350010	2-mercaptoethanol (50 mM)