Generation of Spheroids from Human Pluripotent Stem Cells

Abstract

Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. Vis. Exp. (2022).

This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain organoids.

Protocol

1. Cortical brain organoid generation (Figure 1)

NOTE: All the steps in this section of the protocol will occur in a Class 2 biosafety hood unless stated otherwise.

1. Induction of 2D neuroectodermal colonies from human pluripotent stem cells (hPSCs) 2D culture (Days -1 to 3)
   1. Before induction, plate the hPSC colonies on a human embryonic stem cell (hESC) qualified basement membrane matrix in a 6-well plate at 20%-30% density. Achieve this density by passaging hPSC colonies from one well of a 6-well plate at 60% confluency into three wells of a 6-well plate.
   2. ....

Results

Figure 1: Schematic diagram for generating reproducible cortical brain organoids. Schematic workflow of the experimental procedure for the generation of cortical brain organoids from hPSCs maintained in the feeder-free medium. The workflow provides an ov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Formaldehyde (W/V) Methanol-free	Thermo Fisher Scientific	28908	4% of PFA are diluted in 1x PBS
2-Mercaptoethanol 50 mL(1000x)	Life Technologies Australia (TFS)	21985023	Used in NM and DM media
B 27 Supplement 10 mL	Life Technologies Australia (TFS)	17504044	Used in NM and DM media
CKX53 microscope with SC50 camera	Olympus
Corning Costar 6 well cell culture plates	Sigma Aldrich Pty Ltd	CLS3516-50EA
Dispase II powder	Thermo Fisher Scientific	17105041	Powder is dissolve in HBSS, filtered through 0.22 µm filter, aliquote at 10 mL and store at -20 °C
DMEM Nutrient Mix F12 10x 500 mL (DMEM/F-12)	Thermofisher	11320082	Used in NM and DM media
DMSO Dimethyl Sulfoxide	Sigma Aldrich Pty Ltd	D2650-100ML
Dulbecco's Phosphate Buffered Saline	Sigma Aldrich Pty Ltd	D1408-500ML
Falcon Matrigel hESC-qualified Matrix	In Vitro Technologies Pty Ltd	FAL354277	Make aliquotes of 100 µL and stored at -20 °C
GlutaMAX Supplement 100x	Thermo Fisher Scientific	35050061	Used in NM and DM media
Hanks Balanced Salt Solution	Sigma Aldrich Pty Ltd	H8264
Human induced pluripotent stem cells (EU79)			In-house reporogrammed from skin fibroblast
Human induced pluripotent stem cells (G22)	Genea Biocells		Obtained from Genea Biocells (San Diego, United States)
Human induced pluripotent stem cells (WTC)			Gift from Professor Bruce Conklin
InSolution TGF-Β RI Kinase Inhibitor VI, SB431542	Merck	US1616464-5MG
Insulin Solution Human Recombinant	Sigma Aldrich Pty Ltd	I9278	Used in NM and DM media
LDN193189 Dihydrochloride	Sigma Aldrich Pty Ltd	SML0559-5MG	Used during differentiation
MEM Non-Essential Amino Acids Solution (100x)	Thermo Fisher Scientific	11140050	Used in NM and DM media
mTeSR Plus	STEMCELL TECHNOLOGIES	100-0276	Used to maintain hiPSC colonies prior to differentiation with NM media
N2 Supplement 5 mL (100x)	Life Technologies Australia Pty Ltd	17502048	Used in NM and DM media
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122	Used in NM and DM media
Recombinant Human FGF basic	R&D Systems	233-FB-01M	Aliquots are made at 20 µg/mL and stored at -20 °C
SB431542	Tocris	1614	Used during differentiation
Ultra-Low attachment multiwell plates, 24-well plate, polystyrene	Sigma Aldrich Pty Ltd	CLS3473-24EA