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 sources has been greatly developed in recent years^4,5. Recent publications have shown that human induced pluripotent stem (iPS) cell-derived long-term self-renewing neuroepithelial-like stem (lt-NES) cells, following a cortical differentiation protocol and after intracortical transplantation in a rat model with ischemic stroke affecting the somatosensory cortex, generate mature cortical neurons. In addition, the graft-derived neurons received afferent and efferent synaptic connections from the host neurons, showing their integration into the rat neuronal network^6,7. The graft-derived axons were myelinated and found in different areas of the rat brain, including the peri-infarct area, corpus callosum, and contralateral somatosensory cortex. Most importantly, iPS cell-derived transplantation reversed motor deficits in stroke animals⁷.

Even if animal models help to study transplant survival, neuronal integration, and the effect of the grafted cells on motor and cognitive functions, information about interaction between human cells (graft-host) is missing in this system^8,9. For this reason, a combined method of long-term human brain organotypic culture with the ex vivo transplantation of human iPS cell-derived neuronal progenitors is described here. Human brain organotypic cultures obtained from neurosurgical resections are physiologically relevant 3D models of the brain that allow researchers to increase their understanding of the human central nervous system circuitry and the most accurate way of testing treatments for human brain disorders. However, not enough research has been done in this context, and in most cases, human hippocampal brain organotypic cultures have been used^10,11. The cerebral cortex is affected by several neurodegenerative disorders, such as ischemic stroke¹² or Alzheimer's disease¹³, so it is important to have a human cortical 3D system that allows us to expand our knowledge and to test and validate different therapeutic strategies. Several studies in the last few years have used cultures from adult human cortical (hACtx) tissue to model human brain diseases^{14,15,16,17,18,19}; however, limited information is available in the context of stem cell therapy. Two studies have already demonstrated the feasibility of the system described here. In 2018, human embryonic stem cells programmed with different transcription factors and transplanted into hACtx tissue were shown to give rise to mature cortical neurons that could integrate into adult human cortical networks²⁰. In 2020, the transplantation of lt-NES cells into the human organotypic system revealed their capacity to differentiate into mature, layer-specific cortical neurons with the electrophysiological properties of functional neurons. The grafted neurons established both afferent and efferent synaptic contacts with the human cortical neurons in the adult brain slices, as corroborated by rabies virus retrograde monosynaptic tracing, whole-cell patch-clamp recordings, and immuno-electron microscopy²¹.

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.

1. Tissue collection, maintenance, cutting, and plating

1. Preparations on the day before tissue slicing
   1. Prepare 2 L of cutting solution (Table 1) in a volumetric flask. Dissolve all the ingredients except MgCl₂ and CaCl₂ in ~1,800 mL of deionized water and bubble with carbogen gas for 15 min. Then add appropriate amounts of 1 M MgCl₂ and CaCl₂ solutions and continue bubbling for another 15 min. Finally, fill the flask to the 2 L mark; check the pH and osmolarity and adjust if needed. Freeze 2 x 350 mL of the prepared solution and store the rest at 4 °C.
      NOTE: The pH and osmolarity are typically 7.3-7.4 and 295-300 mOsm, respectively.
   2. Prepare 100 mL of rinsing solution (Table 2). Dissolve 476 mg of HEPES and 200.6 mg of glucose in 100 mL of HBSS supplemented with 5 mL of penicillin/streptomycin. This can be stored at 4 °C for up to 10 days.
   3. Prepare human adult cortical (hACtx) culture medium (hACtx medium) (Table 3) and filter it in the cell culture lab under a ventilated hood. The medium comprises neuronal medium without phenol red (see the Table of Materials), B27 supplement, L-glutamine (see the Table of Materials), and gentamicin. Store at 4 °C for up to 2 weeks.
2. Preparations on the day of the operation before the arrival of the tissue samples
   1. Check the availability of the needed equipment and lab space, and thoroughly clean all the surgical tools and the vibratome (see the Table of Materials), as well as the benchtop space, with distilled water (without detergent), followed by 70% ethanol. Let the ethanol dry for at least 30 min before using the tools and equipment.
   2. Crush the frozen cutting solution and bubble the "soup'' of ice and liquid with carbogen gas for 30 min. Then, tightly close one of the containers with the crushed solution ''soup'' and place it in the icebox. This will be used to collect the tissue from the operation room.
   3. Calibrate the vibratome and set the cutting parameters: 0.05 mm/s speed and 1.7 mm vibration. Place the cutting chamber on a vibratome stage and start the cooler connected to it so that the chamber is at a constant temperature of −3 °C.
   4. Prepare the slice collection chamber with inserts to place the tissue slices and cutting solution, bubbling it constantly with carbogen gas at room temperature (RT).
   5. In the cell culture lab and under a ventilated hood, place the culture inserts in a 6-well plate using forceps. Add 5 mL of hACtx medium on the bottom of the insert until it contacts the membrane, avoiding the formation of bubbles, and add 2 mL on the top of the insert. Equilibrate in the incubator at 37 °C and 5% CO₂ for at least 2 h before transferring the tissue slices into the inserts.
3. Tissue collection and slicing procedures
   1. Immediately after resection, collect the tissue from the patient in the operation room, if possible, directly into a container with frozen, bubbled, and crushed cutting solution. Transfer the closed container on ice immediately to the cutting area of the lab.
   2. Inspect the tissue and locate the best surface to glue (see step 1.3.3) it to the cutting stage of the vibratome, considering the orientation of the cortical layers. If needed, cut the uneven surface with a scalpel so that it is easy to place the tissue on the stage for optimal slice orientation.
   3. Glue the tissue to the stage with tissue adhesive (see the Table of Materials), place it in the slicing chamber, and immediately fill the chamber with cold bubbled cutting solution. Continue the bubbling during the whole cutting procedure.
   4. Cut coronal or sagittal slices, depending on the tissue-to-blade orientation, at a thickness of 300 µm to contain all the cortical layers and, if possible, the white matter. Place the slices in the collection chamber with bubbling cutting solution at RT.
      NOTE: Cut from the white matter side toward the surface of the cortex. Do not remove the meninges, as this may damage the tissue. The cutting blade usually slices through them easily.
   5. Once all the tissue has been cut, transfer the slices into a sterile Petri dish with rinsing solution at RT and transport them to the cell culture lab. This step is necessary to remove excess sucrose from the slices before transferring them to the culture plate.
      NOTE: To transfer the slices (in this and the following steps), use an inverted glass pipette, break off the thinner part, and place a rubber teat for suction.
4. Culture and maintenance of hACtx tissue slices
   1. Individually place the tissue slices on top of the already wet and submerged inserts. After 24 h, change the medium to further remove any remaining sucrose or other residual substances from the cutting procedures.
   2. Replace the culture medium with fresh medium every 7 days. After 2 weeks, use hACtx medium without gentamicin. The culture can be maintained for up to 2 months.
      NOTE: Equilibrate the hACtx medium in the incubator at 37 °C and 5% CO₂ for at least 2 h before the medium change. Fresh medium should be prepared every 2 weeks. Check the slices every 2-3 days and, if some of the medium has evaporated, add more to the top of the insert.

Cutting solution	Stock concentration	Final concentration [mM]	Per 1 L
Sucrose	Powder	200	68.46 g
NaHCO3	Powder	21	1.76 g
KCl	Powder	3	0.22 g
NaH2PO4	Powder	1.25	0.17 g
Glucose	Powder	10	1.80 g
MgSO4	1 M	2	2 mL
CaCl2	1 M	1.6	1.6 mL
MgCl2	2 M	2	1 mL

Table 1: Composition of cutting solution. MgCl₂ and CaCl₂ are used as preprepared 1 M solutions in deionized water.

Rinsing solution	Stock concentration	Final concentration	Per 100 mL
HBSS	1x		95 mL
PenStrep	10,000 U/mL	500 U/mL	5 mL
HEPES	Powder	4.76 g/L	476 mg
Glucose	Powder	2 g/L	200.6 mg

Table 2: Composition of rinsing solution.

hACtx medium	Stock concentration	Final concentration	Per 100 mL
Neuronal medium without Phenol red			97.4 mL	see Table of Materials
B27	50x	1:50	2 mL
L-Glutamine	100x	1:200	500 µL	see Table of Materials
Gentamicin	50 mg/mL	1:1000	100 µL

Table 3: Composition of hACtx medium.

2. Proliferation and differentiation of lt-NES cells

NOTE: Lt-NES cells are generated as previously described^21,22 and transduced with a lentiviral vector carrying green fluorescent protein (GFP) under a constitutive promoter (GFP-lt-NES cells). Vials containing 3 x 10⁶ cells are stored at −150 °C until use.

1. Preparation of the stock solutions and media
   1. Dilute 100 µg of basic fibroblast growth factor (bFGF) in 10 mL of PBS-0.1% BSA to have a stock concentration of 10 µg/mL. Prepare 100 µL aliquots.
   2. Dilute 100 µg of epidermal growth factor (EGF) in 10 mL of PBS-0.1% BSA to have a stock concentration of 10 µg/mL. Prepare 100 µL aliquots.
   3. Aliquot 50x B27 supplement in a volume of 100 µL per aliquot.
   4. Dilute 10 mg of poly-L-ornithine in 100 mL of deionized water to have a stock concentration of 100 µg/mL. Make 1 mL aliquots.
   5. Prepare 30 µL aliquots of 1.20 mg/mL mouse laminin.
   6. Dilute 10 mL of trypsin-EDTA (0.25%) in 90 mL of PBS to a stock concentration of 0.025%. Prepare 1 mL aliquots.
   7. Dilute 0.025 g of trypsin inhibitor in 50 mL of PBS to a stock concentration of 0.5 mg/mL. Prepare 1 mL aliquots.
   8. Dilute 10 µg of Wnt3a (Wingless-type MMTV integration site family member 3A) in 1 mL of PBS-0.1% BSA to have a stock concentration of 10 µg/mL. Prepare BMP4 (bone morphogenetic protein 4) in the same way. Aliquot in a volume of 100 µL.
   9. Dilute 1 mg of cyclopamine in 2.5 mL of DMSO to a final concentration of 400 µg/mL, and make 100 µL aliquots.
   10. Prepare basic medium (Table 4) and differentiation-defined medium (DDM, Table 5), and store at 4 °C for up to 2 weeks.
2. Coating of culture dishes
   1. To pre-coat the dishes to culture the GFP-It-NES cells during proliferation or differentiation, dilute poly-L-ornithine 1:100 in deionized water and add 5 mL of the solution to a T25 flask. Incubate overnight at RT.
   2. Wash the poly-L-ornithine coated plates 1x with deionized water and 1x with PBS.
   3. For GFP-It-NES cells in proliferation, dilute mouse laminin at 1:500 in PBS and incubate for at least 2 h at 37 °C. For GFP-It-NES cells in differentiation, dilute mouse laminin at 1:100 in PBS and incubate for at least 2 h at 37 °C.
3. Proliferation of the GFP-lt-NES cells
   1. Warm 5 mL (for washing) and 5 mL (for seeding) of basic medium in two different 15 mL tubes.
   2. Rapidly thaw one vial of GFP-lt-NES cells at 37 °C, transfer them to the washing tube, and centrifuge at 300 x g for 5 min.
   3. Aspirate the medium carefully without touching the pellet, and resuspend the cells in 1 mL of pre-warmed basic medium. Transfer the cell suspension to the seeding tube containing basic medium supplemented with proliferation factors: EGF (10 ng/mL), bFGF (10 ng/mL), and B27 (10 ng/mL). Seed the cells on a poly-L-ornithine/laminin-coated T25 flask.
   4. Feed the cells with proliferation factors every day. Replace the medium if it turns yellow. Passage the cells every third or fourth day (1 day after they reach 100% confluency).
4. Splitting the GFP-lt-NES cells for proliferation and differentiation
   1. Pre-warm 5 mL of basic medium per flask (to collect the cells), plus the total volume needed to reseed them.
      NOTE: The passage is done at a 1:3 dilution to keep the cells in proliferation, requiring 15 mL of basic medium, and a 1:6 dilution to start differentiation, requiring 30 mL of basic medium.
   2. Remove the medium from the cell culture flask by aspiration, and add 500 µL of pre-warmed 0.025% trypsin. Incubate for 5-10 min at RT. The detachment of the cells can be confirmed under a standard light microscope at 10x magnification.
   3. Add an equal volume of trypsin inhibitor (0.5 mg/mL final concentration) followed by 5 mL of warm basic medium. Detach and collect the cells by gently pipetting up and down. Transfer the cells to a 15 mL tube and centrifuge for 5 min at 300 x g.
   4. To keep the cells in proliferation, replate at a 1:3 dilution in fresh basic medium supplemented with proliferation factors, and repeat step 2.3.4.
5. Cortical differentiation of the GFP-lt-NES cells
   1. On day 0, resuspend the cells (to be used for differentiation) in 30 mL of basic medium supplemented with proliferation factors, and plate them in six differentiation-coated T25 flasks (split 1:6).
   2. On day 1, change half of the medium to DDM, and add proliferation factors at half of their concentration.
   3. On day 2, change the medium completely to DDM supplemented with differentiation factors: BMP4 (10 ng/mL), Wnt3a (10 ng/mL), and cyclopamine (400 ng/mL).
   4. On day 4, add the differentiation factors alone. Replace the medium if it turns yellow.
   5. On day 6, change the medium to DDM supplemented with BMP4 and Wnt3a. The cyclopamine is removed at this step.
   6. On day 7, detach the cells as stated in step 2.4.2 and step 2.4.3.

Basic Medium	Stock concentration	Final concentration	Per 100 mL
DMEM/F12 with L-Glutamine	1x		98.7 mL
N-2 supplement	100x	1:100	1 mL
Glucose	45%	3.5 mL/L	350 µL

Table 4: Composition of proliferation medium of lt-NES cells (basic medium).

DDM medium	Stock concentration	Final concentration	Per 100 mL
DMEM/F12 with L-Glutamine			96 mL
N2	100 x	1:100	1 mL
NEAA	100 x	1:100	1 mL
Sodium Pyruvate	100 mM	1:100	1 mL
BSA V Fraction	7.5%	6.6 mL/L	660 µL
2-mercaptoethanol	50 nM	7 µL/L	0.7 µL
Glucose	45%	3.2 mL/L	320 µL

Table 5: Composition of differentiation-defined medium (DDM) of lt-NES cells.

3. Transplantation of the GFP-lt-NES cells into organotypic hACtx slices

NOTE: The hACtx tissue should be cultured for 1 week prior to cell transplantation. To facilitate the transplantation procedure, it is necessary to remove 2 mL of the hACtx medium from the top of the insert to prevent the tissue from floating.

1. Resuspend the cortically primed GFP-lt-NES cells (from step 2.5.6) in cold pure basement membrane matrix (see the Table of Materials) at a concentration of 1 x 10⁵ cells/µL and transfer the solution to a smaller sterile tube.
   NOTE: During the transplantation procedure, all the materials (pipette tips, tubes, capillary, etc.) should be pre-cooled to avoid gel solidification. Thaw the basement membrane matrix gel on ice for 30 min before its use.
2. Collect the cell suspension into a cold glass capillary connected to a rubber teat for suction. Inject the cell suspension as small drops (approx. 1 µL each) by stabbing the semi-dry tissue slice at various sites.
3. Incubate at 37 °C for 30 min for the gel to solidify. Transfer the plate from the incubator back to the hood, and carefully add 2 mL of hACtx medium to the top of the insert to completely submerge the tissue.
4. Replace the culture medium with fresh hACtx medium once per week.

4. Validation

1. Staining of the hACtx slices
   1. At the desired time point, take out the slices from the cell culture lab, and remove them from the insert by immersing it in a Petri dish with PBS. Then, transfer the slices to staining vials using an inverted glass pipette (see the NOTE in step 1.3.5), and fix them with 4% paraformaldehyde (PFA) overnight at 4 °C.
   2. Rinse 3x with KPBS for 15 min each time, and incubate overnight at 4 °C with permeabilization solution (0.02% BSA and 1% Triton X-100 in KPBS).
   3. The next day, add blocking solution (KPBS with 0.2% Triton X-100, 1% BSA, sodium azide [1:10,000], and 10% normal donkey serum), and incubate overnight at 4 °C.
   4. After blocking, add the primary antibodies (see Table 6 for the dilutions) diluted in the blocking solution, and incubate for 48 h at 4 °C.
   5. Wash 3x with blocking solution without adding serum for 15 min each. Add the secondary antibodies diluted in blocking solution (see Table 6 for the dilutions), and incubate for 48 h at 4 °C.
   6. Wash 3x with blocking solution without serum, and incubate for 2 h at RT in Hoechst stain diluted in permeabilization solution (1:1,000).
   7. Wash 3x with KPBS, mount the slices on glass slides using a paintbrush, and let them dry. Finally, rinse the slides with deionized water, remove excess water, add the mounting medium, and cover with a glass coverslip. Keep the slides for at least 24 h at RT, and store them at 4 °C until imaging.
      NOTE: For antibodies labeling nuclear epitopes, perform antigen retrieval prior to permeabilization (step 4.1.2) with sodium citrate (10 mM, pH 6.0) for 2 h at 65 °C.
2. Whole-cell patch-clamp
   1. On the day of recording, prepare 1 L of human artificial cerebrospinal fluid (haCSF), modified to better match the human brain environment (Table 7). Similar to the cutting solution, make approximately 900 mL of the solution with all the ingredients except CaCl₂ dissolved in deionized water, and bubble it with carbogen for 15 min before adding the appropriate volume of 1 M CaCl₂ solution. Fill up the volumetric flask to the 1 L mark, and continue bubbling at RT for an additional 15 min before starting the recording and throughout the experiment.
   2. Transfer the tissue slices from the culture plate to the recording chamber on the stage of an upright microscope that is constantly perfused with bubbled haCSF at a perfusion rate of 2 mL/min and warmed to 34 °C using a bath temperature controller.
   3. Pull glass capillaries with a pipette puller to an average resistance of 3-5 MΩ, and backfill the capillaries with K-gluconate-based internal solution (Table 8) with a freshly added 2-4 mg of biocytin for the post-hoc identification of the recorded cells. The pH and osmolarity of this internal solution are 7.2-7.3 and 285-295 mOsm, respectively.
   4. In the case of host cell recording, get a rough overview of the slice with a 4x objective, and find a healthy-looking cell to patch with a 40x objective. Then, proceed with the standard whole-cell patch clamp.
   5. In the case of grafted cell recording, identify the tissue area with the grafted cells using a 4x objective and an epifluorescence filter in the blue range (460 nm) by GFP reporter expression in the graft. Then, zoom in to the located area with a 40x objective, and find a grafted cell expressing GFP for a standard whole-cell patch clamp.
   6. Check the resting membrane potential (RMP) immediately after breaking into the cell, and make sure that the quality of the recording is good. Record all the parameters of interest (e.g., membrane resistance [Ri], AP parameters, sodium and potassium currents, and synaptic activity) in the whole-cell voltage or current-clamp configuration.
   7. After collecting all the necessary data, carefully retract the recording pipette without further damaging the cell so that it can be identified with post-hoc immunostaining.
   8. Transfer the slice to the 4% PFA solution for further fixation and staining, as described in step 4.1. Streptavidin is used to immunolabel the cells filled with biocytin during the recordings.

ANTIBODIES	Dilution	Notes
Primary
Chicken anti-GFP	1:1000
Chicken anti-MAP2	1:1000
Goat anti-AiF1	1:100
Mouse anti-MBP	1:1000	Antigen retrieval needed
Mouse anti-SC123	1:2000
Rabbit anti-NeuN	1:1000
Rabbit anti-Olig2	1:500
Rabbit anti-Tmem119	1:200
Secondary
488-conjugated AffinityPure Donkey anti-mouse IgG	1:500
488-conjugated AffinityPure Donkey anti-rabbit IgG	1:500
488-conjugated AffinityPure Donkey anti-chicken IgG	1:500
Cy3-conjugated AffinityPure Donkey anti-chicken IgG	1:500
Cy3-conjugated AffinityPure Donkey anti-goat IgG	1:500
Cy3-conjugated AffinityPure Donkey anti-mouse IgG	1:500
Alexa fluor 647-conjugated Streptavidin	1:500

Table 6: List of primary and secondary antibodies for immunohistochemistry.

haCSF	Stock concentration	Final concentration [mM]	Per 1 L
NaCl	Powder	129	7.54 g
NaHCO3	Powder	21	1.76 g
Glucose	Powder	10	1.80 g
KCl	Powder	3	0.22 g
NaH2PO4	Powder	1.25	0.17 g
MgSO4	1 M	2	2 mL
CaCl2	1 M	1.6	1.6 mL

Table 7: Composition of artificial cerebrospinal fluid (haCSF).

K-Gluconate internal solution	Stock concentration	Final concentration [mM]	Per 100 mL
K-gluconate	Powder	122.5	2.87 g
KCl	Powder	12.5	93.18 mg
NaCl	Powder	8	46.76 mg
HEPES	Powder	10	238.32 mg
MgATP	Powder	2	101.4 mg
Na3GTP	Powder	0.3	17.0 mg
Note: Adjust pH with KOH/HCl

Table 8: Composition of K-gluconate-based internal solution.

Results

Following the described protocol, hACtx tissue from a patient with temporal lobe epilepsy was collected and processed, as explained above. A few slices were fixed after 24 h in culture to study the starting point of the host tissue. The analysis of different neural cell populations such as neurons (expressing NeuN and Map2, Figure 1A), oligodendrocytes (Olig2 and MBP, Figure 1B), and astrocytes (human-specific GFAP, also named STEM123, Figur...

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