Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Summary

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Abstract

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Introduction

Neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, or ischemic stroke, are a group of diseases that share the common feature of neuronal malfunction or death. They are heterogeneous in terms of the brain area and neuronal population affected. Unfortunately, treatments for these diseases are scarce or of limited efficacy due to the lack of animal models that mimic what occurs in the human brain^1,2. Stem cell therapy is one of the most promising strategies for brain regeneration³. The generation of neuronal progenitors from stem cells from different sou....

Protocol

This protocol follows the guidelines approved by the Regional Ethical Committee, Lund, Sweden (ethical permit number 2021-07006-01). Healthy neocortical tissue was obtained from patients undergoing elective surgery for temporal lobe epilepsy. Informed consent was obtained from all patients.

NOTE: All the tissues obtained were processed regardless of their size. However, tissues smaller than 1-1.5 mm³ in size will be technically challenging to handle and section with a vibratome.

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Discussion

Obtaining hACtx slices of high enough quality is the most critical step in this protocol. Cortical tissue is obtained from epileptic patients undergoing resective surgery²⁴. The quality of the resected tissue, as well as the exposure time of the tissue between resection and culture, is critical; the faster the tissue is transferred from the surgery room to the laboratory and cut, the more optimal the organotypic culture will be. Ideally, the tissue should be cut and transferred to.......

Materials

Name	Company	Catalog Number	Comments
Tissue Cutting and electrophysiology
Adenosine 5'-triphosphate magnesium salt	Sigma	A9187
Bath temperature controller	Luigs & Neumann	TC0511354
Calcium Chloride dihydrate	Merck	102382
Carbogen gas	Air Liquide	NA
Cooler	Julaba FL 300	9661012.03
D-(+)Glucose	Sigma-Aldrich	G7021
Double Patch-Clamp amplifier	HEKA electronic	EPC10
Guanosine 5'-Triphosphate disodium salt	Millipore	371701
HEPES	AppliChem	A1069
Magnesium Chloride hexahydrate	Sigma-Aldrich	M2670
Magnesium Sulfate heptahydrate	Sigma-Aldrich	230391
Patchmaster	HEKA electronic	Patchmaster 2x91
Pipette Puller	Sutter	P-2000
Plastic Petri dish	Any suitable
Potassium chloride	Merck	104936
Potassium D-gluconate	ThermoFisher	B25135
Rubber teat + glass pipette	Any suitable
Sodium Bicarbonate	Sigma-Aldrich	S5761
Sodium Chloride	Sigma-Aldrich	S7653
Sodium dihydrogen phosphate monohydrate	Merck	106346
Sucrose	Sigma-Aldrich	S7903
Tissue adhesive: Acryl super glue	Loctite	2062278
Upright microscope	Olympus	BX51WI
Vibratome	Leica	VT1200 S
RINSING SOLUTION
D-(+)Glucose	Sigma-Aldrich	G7021
HBSS (without Ca, Mg, or PhenolRed)	ThermoFisher Scientific	14175095
HEPES	AppliChem	A1069
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15-140-122
MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE
6-well plate	ThermoFisher Scientific	140675
Alvetex scaffold 6 well insert	Reinnervate Ltd	AVP004-96
B27 Supplement (50x)	ThermoFisher Scientific	17504001
BrainPhys without Phenol Red	StemCell technologies	#05791	Referenced as neuronal medium in the text
Filter units 250 mL or 500 mL	Corning Sigma	CLS431096/97
Forceps	Any suitable
Gentamicin (50 mg/mL)	ThermoFisher Scientific	15750037
Glutamax Supplement (100x)	ThermoFisher Scientific	35050061	Referenced as L-glutamine in the text
Rubber teat + Glass pipette	Any suitable
GENERATION OF lt-NES cells
2-Mercaptoethanol 50 mM	ThermoFisher Scientific	31350010
Animal Free Recombinant EGF	Peprotech	AF-100-15
B27 Suplemment (50x)	Thermo Fisher Scientific	17504001
bFGF	Peprotech	AF-100-18B
Bovine Albumin Fraction V (7.5% solution)	ThermoFisher Scientific	15260037
Cyclopamine, V. calcifornicum	Calbiochem	# 239803
D (+) Glucose solution (45%)	Sigma	G8769
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	D2438-10mL
DMEM/F12	ThermoFisher Scientific	11320074
Dulbecco's Phosphate Buffer Saline (DPBS)	Thermo Fisher Scientific	14190-144	Without calcium and magnesium
Laminin Mouse Protein, Natural	Thermo Fisher Scientific	23017015
MEM Non-essential aminoacids solutions (100x)	ThermoFisher Scientific	11140050
N-2 Supplement (100 x)	ThermoFisher Scientific	17502001
Poly-L-Ornithine	Merk	P3655
Recombinant Human BMP-4 Protein	R&D Systems	314-BP-010
Recombinant Human Wnt-3a Protein	R&D Systems	5036-WN
Sodium Pyruvate (100 mM)	ThermoFisher Scientific	11360070
Soybean Trypsin Inhibitor, powder	Thermo Fisher Scientific	17075029
Sterile deionized water	MilliQ		MilliQ filter system
Trypsin EDTA (0.25%)	Sigma	T4049-500ML
EQUIPMENT FOR CELL CULTURE
Adjustable volume pipettes 10, 100, 200, 1000 µL	Eppendorf	Various
Basement membrane matrix ESC-qualified (Matrigel)	Corning	CLS354277-1EA
Centrifuge	Hettich Centrifugen	Rotina 420R	5% CO2, 37 °C
Incubator	ThermoForma Steri-Cult CO2	HEPA Class100
Stem cell cutting tool 0.190-0.210 mm	Vitrolife	14601
Sterile tubes	Sarstedt	Various
Sterile Disposable Glass Pasteur Pipettes 150 mm	VWR	612-1701
Sterile pipette tips 0.1-1000  µL	Biotix VWR	Various
Sterile Serological Pipettes 5, 10, 25, 50 mL	Costar	Various
T25 flasks Nunc	ThermoFisher Scientific	156367
IMMUNOHISTOCHEMISTRY
488-conjugated AffinityPure Donkey anti-mouse IgG	Jackson ImmunoReserach	715-545-151
488-conjugated AffinityPure Donkey anti-rabbit IgG	Jackson ImmunoReserach	711-545-152
488-conjugated AffinityPure Donkey anti-chicken IgG	Jackson ImmunoReserach	703-545-155
Alexa fluor 647-conjugated Streptavidin	Jackson ImmunoReserach	016-600-084
Bovine Serum Albumin	Jackson ImmunoReserach	001-000-162
Chicken anti-GFP	Merk Millipore	AB16901
Chicken anti-MAP2	Abcam	ab5392
Cy3-conjugated AffinityPure Donkey anti-chicken IgG	Jackson ImmunoReserach	703-165-155
Cy3-conjugated AffinityPure Donkey anti-goat IgG	Jackson ImmunoReserach	705-165-147
Cy3-conjugated AffinityPure Donkey anti-mouse IgG	Jackson ImmunoReserach	715-165-151
Diazabicyclooctane (DABCO)	Sigma Aldrich	D27802	Mounting media
Goat anti-AIF1 (C-terminal)	Biorad	AHP2024
Hoechst 33342	Molecular Probes		Nuclear staining
Mouse anti-MBP	BioLegend	808402
Mouse anti-SC123	Stem Cells Inc	AB-123-U-050
Normal Donkey Serum	Merk Millipore	S30-100
Paint brush	Any suitable
Paraformaldehyde (PFA)	Sigma Aldrich	150127
Potassium Phospate Buffer Saline, KPBS (1x)
Distilled water
Potassium dihydrogen Phospate (KH2PO4)	Merk Millipore	104873
Potassium phospate dibasic (K2HPO4)	Sigma Aldrich	P3786
Sodium chloride (NaCl)	Sigma Aldrich	S3014
Rabbit anti-NeuN	Abcam	ab104225
Rabbit anti-Olig2	Abcam	ab109186
Rabbit anti-TMEM119	Abcam	ab185333
Sodium azide	Sigma Aldrich	S2002-5G
Sodium citrate
Distilled water
Tri-Sodium Citrate	Sigma Aldrich	S1804-500G
Tween-20	Sigma Aldrich	P1379
Triton X-100	ThermoFisher Scientific	327371000
EQUIPMENT FOR IMMUNOHISTOCHEMISTRY
Confocal microscope	Zeiss	LSM 780
Microscope Slides 76 mm x 26 mm	VWR	630-1985
Microscope Coverslips 24 mm x 60 mm	Marienfeld	107242
Microscope Software	Zeiss	ZEN Black edition
Rubber teat + Glass pipette	Any suitable