Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Abstract

Source: Koch, L. S., et al. Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research. J. Vis. Exp. (2023).

This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

Protocol

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

Figure 1 illustrates a schematic overview of the workflow of this protocol.

1. iPSC reprogramming and maintenance

1. Collect 8 mL of the subject's blood (aged or disease patient; ages 65 and older) into cell preparation tubes (CPT) with sodium citrate or into ethylenediaminetetraacetic acid (EDTA) or heparinized tubes.
2. Centrifuge at 1,800 x g for 30 min at room temperature (RT) to collect pellet containing only peripheral blood and no serum....

Results

Figure 1: Schematic illustration of the workflow and timeline of this method.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-β-Mercaptoethanol	Thermo Fisher Scientific	31350010
4′,6-Diamidino-2-phenylindoledihydrochloride (DAPI)	Invitrogen	D1306	67:40:00
6-well Clear Flat Bottom CELLSTAR Cell Culture Multiwell Plate	Greiner Bio-One	657185
6-well Clear Flat Bottom Ultra-Low Attachment Well Plate	Corning	3471
96-well Clear Round Bottom Ultra-Low Attachment Microplate	Corning	7007
96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate	Thermo Fisher Scientific	136101
Accutase	Sigma-Aldrich	46964	cell detachment solution
Antibiotic-Antimycotic (100x)	Gibco	15240062
B27 Suppement (with Vitamin A) (50x)	Gibco	17504044
B27 Supplement (minus Vitamin A) (50x)	Gibco	12587010
BD Vacutainer™ Glass Mononuclear Cell Preparation (CPT) Tubes	Thermo Fisher Scientific	02-685-125
Bovine Serum Albumin	Sigma-Aldrich	A9418
Centrifuge	Eppendorf	5810 R	With plate holders
CHIR99021	Selleck Chemicals	S2924
CytoTune Sendai Reprogramming Vector	Thermo Fisher Scientific	A1378001
ddPCR primers | human | MAPT	Bio-Rad	dHsaCPE192234
ddPCR primers | human | RBFOX3 (NeuN)	Bio-Rad	dHsaCPE5052108
DMEM/F12	Thermo Fisher Scientific	11320074
Doublecortin (DCX)	Santa Cruz Biotechnology	SC-8066	09:20
Dulbecco's phosphate buffered saline (DPBS)	Thermo Fisher Scientific	14190144	no calcium, no magnesium
Eppendorf cups, 1.5 mL	Eppendorf	0030 125.215
Essential 6	Gibco	A1516401
Essential 8	Gibco	A1517001
Ethylenediaminetetraacetic acid (EDTA)	Invitrogen	15575020
Falcon tubes, 15 mL, conical	Greiner Bio-One	188271-N
Formaldehyde	Sigma-Aldrich	252549	37% Stock solution, diluted to 4% in PBS
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix	Thermo Fisher Scientific	A1413202
GlutaMax (100x)	Gibco	35050038
Hemacytometer cell counter	Hausser scientific	1490
HEPES Buffer	Thermo Fisher Scientific	15-630-080
Insulin	Sigma-Aldrich	I9278
LDN 193189	StemCell Technologies	72147
MAP2	Abcam	ab32454	04:20
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free	Corning	356230	basement membrane matrix
MEM-Non Essential Amino Acid Solution (MEM-NEAA; 100x)	Thermo Fisher Scientific	11140050
Multilabel Counter Victor 3 Plate Reader	Perkin Elmer	1420	luminometer
MycoAlert Mycoplasma Detection Kit	Lonza	LT07-318
N-2 Supplement (100x)	Thermo Fisher Scientific	17502-048
NeuN	Millipore	MAB377	09:20
Neurobasal Medium	Thermo Fisher Scientific	21103049
Oct-3/4 Antibody (C-10) Alexa Fluor 647	Santa Cruz Biotechnology	sc-5279 AF647	02:40
Parafilm	Bemis	PM-996	thermoplastic film sheet
PAX6	Thermo Fisher Scientific	42-6600	04:20
Penicillin/Streptomycin	Gibco	15070063
Poly(lactic-co-glycolic acid) (PLGA) microfilaments	Ethicon	J463
QX200 Droplet digital PCR system	Bio-Rad	1864001
ROCK inhibitor (Y27632)	Selleck Chemicals	S1049
SB431542	R&D Systems	1614/50
SOX2 Monoclonal Antibody (Btjce), Alexa Fluor 488, eBioscience	Invitrogen	53-9811-80	02:40
Synapsin I (SYN)	Calbiochem	574777	04:20
Triton-X 100	Sigma-Aldrich	T8787
TUJ1	Santa Cruz Biotechnology	sc-80005	Beta-3-tubulin; 1:500
XAV939	Tocris Bioscience	3748