Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Summary

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Abstract

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

Introduction

The emergence of human pluripotent stem cells (PSCs) represents a excellent tool for regenerative medicine and disease modeling, because these cells can be differentiated into most cell lineages of the human body^1,2. Since their discovery, PSC differentiation using diverse approaches has been reported to model different diseases, including neurodegenerative disorders^3,4,5,6.

Recently, there have been reports of 3D cultures derived from PSCs resembling huma....

Protocol

1. Passaging and maintenance of human iPSCs in monolayer culture

1. Preparation of plates
   1. Thaw the basement membrane matrix (see Table of Materials) stock at 4 °C and prepare 60 μL aliquots. Freeze the aliquots at -20 °C.
   2. To coat the wells of a 6 well plate, thaw one aliquot of the basement membrane matrix on ice. Once thawed add 60 µL to 6 mL of DMEM-F12. Gently resuspend by pipetting up and down.
   3. Add 1 mL of diluted basement membrane matrix.......

Discussion

The need for large cell numbers as well as defined culture conditions to generate specific cell types for drug screening and regenerative medicine applications has been driving the development of scalable culture systems. In recent years, several groups have reported the scalable generation of neural progenitors and functional neurons^32,33,34, providing significant advances in the development of new models for neurodegenerative .......

Materials

Name	Company	Catalog Number	Comments
3MM paper	WHA3030861	Merck
Accutase	A6964 - 500mL	Sigma	cell detachment medium
Anti-BARHL1 Antibody	HPA004809	Atlas Antibodies
Anti-Calbindin D-28k Antibody	CB28	Millipore
Anti-MAP2 Antibody	M4403	Sigma
Anti-N-Cadherin Antibody	610921	BD Transduction
Anti-NESTIN Antibody	MAB1259-SP	R&D
Anti-OLIG2 Antibody	MABN50	Millipore
Anti-PAX6 Antibody	PRB-278P	Covance
Anti-SOX2 Antibody	MAB2018	R&D
Anti-TBR1 Antibody	AB2261	Millipore
Anti-TBR2 Antibody	ab183991	Abcam
Anti-TUJ1 Antibody	801213	Biolegend
Apo-transferrin	T1147	Sigma
BrainPhys Neuronal Medium N2-A & SM1 Kit	5793 - 500mL	Stem cell tecnhnologies
Chemically defined lipid concentrate	11905031	ThermoFisher
Coverslips 24x60mm	631-1575	VWR
Crystallization-purified BSA	5470	Sigma
DAPI	10236276001	Sigma
Dibutyryl cAMP	SC- 201567B -500mg	Frilabo
DMEM-F12	32500-035	ThermoFisher
Fetal bovine serum	A3840001	ThermoFisher
Gelatin from bovine skin	G9391	Sigma
Glass Copling Jar	E94	ThermoFisher
Glutamax I	10566-016	ThermoFisher
Glycine	MB014001	NZYtech
Ham’s F12	21765029	ThermoFisher
Human Episomal iPSC Line	A18945	ThermoFisher	iPSC6.2
IMDM	12440046	ThermoFisher
Insulin	91077C	Sigma
iPS DF6-9-9T.B		WiCell
Iso-pentane	PHR1661-2ML	Sigma
L-Ascorbic acid	A-92902	Sigma
Matrigel	354230	Corning	basement membrane matrix
Monothioglycerol	M6154	Sigma
Mowiol	475904	Millipore	mounting medium
mTeSR1	85850 -500ml	Stem cell technologies
N2 supplement	17502048	ThermoFisher
Neurobasal	12348017	ThermoFisher
Paraformaldehyde	158127	Sigma
PBS-0.1 Single-Use Vessel	SKU: IA-0.1-D-001	PBS Biotech
PBS-MINI MagDrive Base Unit	SKU: IA-UNI-B-501	PBS Biotech
Recombinant human BDNF	450-02	Peprotech
Recombinant human bFGF/FGF2	100-18B	Peprotech
Recombinant human FGF19	100-32	Peprotech
Recombinant human GDNF	450-10	Peprotech
Recombinant human SDF1	300-28A	Peprotech
ROCK inhibitor Y-27632	72302	Stem cell technologies
SB431542	S4317	Sigma
Sucrose	S7903	Sigma
SuperFrost Microscope slides	12372098	ThermoFisher	adhesion microscope slides
Tissue-Tek O.C.T. Compound	25608-930	VWR
Tris-HCL 1M	T3038-1L	Sigma
Triton X-100	9002-93-1	Sigma
Tween-20	P1379	Sigma
UltraPure 0.5M EDTA, pH 8.0	15575020	ThermoFisher