May 27, 2020

Public workspaceGeneration and assembly of human brain region-specific organoids

This protocol is a draft, published without a DOI.
Generation and assembly of human brain region-specific organoids
  • Steven A. Sloan1,
  • Jimena Anderson1,
  • Anca M. Pașca2,
  • Fikri Birey1,
  • Sergiu Pasca
  • 1Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.;
  • 2Division of Neonatology, Department of Pediatrics, Stanford University, Stanford, CA, USA.
  • Neurodegeneration Method Development Community
  • Organoid and Assembloid
Sergiu Pasca
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External link: https://www.nature.com/articles/s41596-018-0032-7#article-info
Protocol Citation: Steven A. Sloan, Jimena Anderson, Anca M. Pașca, Fikri Birey, Sergiu Pasca 2020. Generation and assembly of human brain region-specific organoids. protocols.io https://protocols.io/view/generation-and-assembly-of-human-brain-region-spec-4wtgxen
Manuscript citation:
Sloan, S.A., Andersen, J., Pașca, A.M. et al. Generation and assembly of human brain region–specific three-dimensional cultures. Nat Protoc 13, 2062–2085 (2018). https://doi.org/10.1038/s41596-018-0032-7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2019
Last Modified: May 27, 2020
Protocol Integer ID: 25267
Keywords: Three-dimensional Culture, 3D Culture, Brain, Human, hCSs, hPSCs, hSSs, initial generation of neural spheroid, establishing human cortical spheroid, human cortical spheroid, neural spheroid, specific forebrain spheroid, forebrain spheroid, human subpallial spheroid, assembly of human brain region, specific spheroid, spheroid, development of the human nervous system, human brain development, dorsal forebrain, ventral forebrain fate, human brain region, astrocyte, human pluripotent stem cell, brain tissue, human forebrain, specific aspects of the human brain, brain region, diseased human brain tissue, gabaergic neuron, human nervous system, human brain, present in the human forebrain, features of human cns development, specific organoid, specific organoids the ability, brain, human cns development, nervous system, challenging ongoing endeavors in neurobiology, brain disorder, cellular study, neurobiology, cell imaging, initial generation, cell, vivo
Abstract
The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region–specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain–specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region–specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.


Introduction

Understanding the development of the human nervous system and elucidating the mechanisms that lead to brain disorders represent some of the most challenging ongoing endeavors in neurobiology. One major obstacle is the restricted access to healthy and diseased human brain tissue for functional molecular and cellular studies. As a result, experimental paradigms have been largely confined to animal models or in vitro cell culture systems that do not fully recapitulate the developmental, architectural, and species-specific aspects of the human brain. Thus, there is a great need for humanderived model systems that recapitulate features of human CNS development and allow for the study of these processes in both healthy and diseased conditions.

Guidelines
Experimental design

Before beginning experiments, it is important to consider several key points that will help determine the scale and time required to perform adequately powered differentiation experiments:

● All experiments should be performed using at least three hPSC lines, with at least three separate differentiations, and using at least two to three neural spheroids per differentiation or condition.
● If validating disease phenotypes, the use of isogenic hPSC lines is essential.

● The results from hESC and hiPSC experiments should be reported separately. We have successfully generated neural spheroids from >70 hiPSC lines and hESCs such as H9.

● Some line-to-line heterogeneity (especially in early stages of differentiation) can be expected, but there should be consistency within lines and across separate differentiations of the same line.

● For intra-spheroid quantifications, at least three to five sections per spheroid should be used. In addition, it is important to undertake the following quality-control steps during the differentiation and patterning process:

● Cultures of hPSCs, hCSs, and hSSs must be regularly checked for mycoplasma (one to two times/ month).

● Genome integrity of hPSCs must be verified regularly by SNP array or comparative genomic hybridization arrays to identify de novo genomic events. If any abnormalities are noted, earlier passages of cells should be used. For differentiations, we primarily use hPSC lines between passages 10 and 40.

● The quality (size, shape, tendency to differentiate) of hPCSs must be checked daily before starting a new differentiation experiment. It is important to ensure that the properties of hPSCs are consistent across passages and before differentiations.

● Cultures should be checked regularly for bacterial/fungal infection. If the medium is found to be cloudy, it should be checked under the microscope at 40× magnification to identify moving bacteria or fungi. It is important to determine the type of contamination immediately and take appropriate measures.

● From days 0 to 6 of differentiation, newly formed neural spheroids must be checked daily for disintegrating/dying cells (the medium will be cloudy at the bottom of the plate and many floating single cells may be observed under the microscope). If cell death or spheroid disintegration exceeds expectations, it is likely that the differentiation will not progress, and it is better to discard the plates.

● The health of spheroids may vary over the culture period and be dependent on the quality of the founder hPSC colonies. Consider sampling hCSs or hSSs and carrying out periodic immunostaining to assess health. Generally, autofluorescence in the center of sectioned spheroids or detaching cells on the surface indicates cell death.
● Consider introducing quality controls for differentiation efficiency by running quantitative PCRs (qPCRs) for a panel of fate- and region-specific genes at particular time points during the differentiation (e.g., day 25).
Materials
Biological materials

  • Human pluripotent stem cells (hPSCs); forebrain spheroids can be generated from cultures of either hESCs, e.g., H9, or hiPSCs. ! CAUTION The experiments involving hPSCs in this study were approved by the Stanford University School of Medicine Institutional Review Board (IRB) and Stem Cell Research Oversight. The hPSCs used to generate the results shown in this protocol were derived at Stanford University and validated using standardized methods (e.g., pluripotency assays, genomeintegrity checks). Cultures were tested and maintained mycoplasmafree. All experiments involving the use of hPSCs must conform to institutional and federal regulations, including IRB panel approval and appropriate informed consent.
  • EmbryoMax primary mouse embryo fibroblasts, neo resistant, Myto-C treated (Millipore, cat. no. PMEF-N-K); alternatively, DR4 mouse embryo fibroblasts (Stanford Human Pluripotent Stem Cells Core Facility) can also be used.


Reagents

Media and supplements

  • DMEM (Life Technologies, cat. no. 10313-039)
  • DMEM/F12 (1:1; Life Technologies, cat. no. 11330-032)
  • Neurobasal A medium (Life Technologies, cat. no. 10888022) ! CAUTION Neurobasal A medium is extremely sensitive to changes in temperature and light. Keep refrigerated at all times.
  • FBS albumin, certified (Life Technologies, cat. no. 16000-044) ! CRITICAL To avoid lot-to-lot variability biases, we recommend testing and purchasing this product in bulk.
  • KnockOut serum replacement (KSR; Life Technologies, cat. no. 10828-028) ! CRITICAL To avoid lot-to-lot variability biases, we recommend testing and purchasing this product in bulk.
  • B27 supplement (Life Technologies, cat. no. 12587010) ! CRITICAL Use B27 supplement without vitamin A. To avoid lot-to-lot variability biases, we recommend testing and purchasing this product in bulk.
  • GlutaMAX supplement, 200 mM (Life Technologies, cat. no. 35050-061)
  • MEM non-essential amino acids (NEAA) solution, 100× (Life Technologies, cat. no. 11140-050)
  • Penicillin–streptomycin (Pen–Strep), 10,000 U/ml (Life Technologies, cat. no. 15070-063) ! CRITICAL Differentiations can also be performed without Pen–Strep.
  • 2-Mercaptoethanol (Sigma-Aldrich, cat. no. M3148)
  • Human recombinant FGF2 (R&D Systems, cat. no. 233-FB)
  • Rock inhibitor Y-27632 (Selleckchem, cat. no. S1049)
  • SMAD inhibitor dorsomorphin (DM; AKA compound C; Sigma-Aldrich, cat. no. P5499)
  • SMAD inhibitor SB-431542 (SB; R&D Systems/Tocris, cat. no. 1614)
  • Human recombinant EGF (R&D Systems, cat. no. 236-EG)
  • Wnt inhibitor (IWP-2; Selleckchem, cat. no. S7085)
  • Smoothened agonist (SAG; Selleckchem, cat. no. S7779)
  • Allopregnanolone (AlloP; Cayman Chemicals, cat. no. 16930)
  • Retinoic acid (RA; Sigma-Aldrich, cat. no. R2625)
  • Human recombinant brain-derived neurotrophic factor (BDNF; PeproTech, cat. no. 450-02)
  • Human recombinant neurotrophin 3 (NT3; PeproTech, cat. no. 450-03)
  • 30% (wt/vol) D(+)-Glucose (Sigma, cat. no. G7021)
  • NaHCO3 (Sigma, cat. no. S5761)
  • EDTA (Sigma, cat. no. ED)

Cell culture reagents

  • Gelatin solution bioreagent, 2% in H2O (Sigma, cat. no. G1393)
  • Dispase (Invitrogen, cat. no. 17105-041)
  • Dulbecco’s DPBS with calcium and magnesium (DPBS; Caisson Labs, cat. no. PBL02)
  • Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. D2650)
  • Ethyl ethanol, 200 proof (Gold Shield, cat. no. 412804) ! CAUTION Ethyl ethanol is flammable and should be maintained in a flame-protective cabinet.

For single-cell dissociation only

  • 10× Earle’s Balanced Salt Solution (EBSS; Sigma, cat. no. E7510)
  • BSA (Sigma, cat. no. A4161)
  • 0.4% (vol/vol) DNase, 12,500 U/ml (Worthington, cat. no. LS002007)
  • L-Cysteine hydrochloride monochloride (Sigma, cat. no. C7880)
  • Papain (Worthington, cat. no. LS 03126)
  • Trypsin inhibitor (Worthington, cat. no. LS003086)
  • Low-Ovo solution (10×) (Reagent setup)
  • High-Ovo solution (10×) (Reagent setup)

For cryosectioning and immunostaining only

  • 16% (vol/vol) Paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15710) ! CAUTION PFA must be handled in a safety cabinet and should be disposed of according to institutional guidelines.
  • Sucrose (Sigma-Aldrich, cat. no. S9378)
  • Fisher Healthcare Tissue-Plus OCT Compound, clear (Fisher Scientific, cat. no. 23-730-571)
  • Triton X-100, laboratory grade (Sigma-Aldrich, cat. no. X100)
  • Normal donkey serum (NDS; EMD Millipore, cat. no. S30-M)
  • Primary antibodies (Table 1)
  • Secondary antibodies, species-specific anti-IgG (H+L), Alexa Fluor–conjugated (Molecular Probes and Jackson Immunoresearch)
  • Hoechst 33258, pentahydrate (bis-benzimide; Life Technologies, cat. no. H3569)
  • Aqua-Poly/Mount (Polysciences, cat. no. 18606)

For optical clearing of intact spheroids by immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) only

  • Methanol (certified ACS; Fisher Chemical, cat. no. A412-1)
  • Hydrogen peroxide, H2O2 (certified ACS; Fisher Chemical, cat. no. H325-100)
  • Glycine (Sigma-Aldrich, cat. no. G7126)
  • Goat serum, heat-inactivated (MP Biomedicals, cat. no. 092939249)
  • Heparin sodium salt (Sigma-Aldrich, cat. no. 84020)
  • Tween-20 (Sigma-Aldrich, cat. no. P1379)
  • Tetrahydrofuran, anhydrous (THF; Sigma-Aldrich, cat. no. 186562)
  • Dichloromethane (Sigma-Aldrich, cat. no. 270997)
  • Benzyl ether (Sigma-Aldrich, cat. no. 108014)
  • Anti-GFP (GeneText, GTX113617)
  • RS Hughes RTV108, 2.8-oz. silicone sealant (Fisher Scientific, cat. no. NC0380109)

For calcium imaging only

  • Fluo-4 acetoxymethyl ester (Fluo-4AM; Invitrogen, F14201)


Equipment

  • Primaria cell culture dish, 10 cm (BD Falcon, cat. no. 353803)
  • Sterile plastic tubes, 15 ml and 50 ml (Corning, cat. no. 430791 and 430829)
  • Ultra-low-attachment plates (Corning, cat. no. 3262 (10 cm), 3261 (6 cm), 3471 (six well), and 3473 (24 well))
  • Fisherbrand premium microcentrifuge tubes, natural, 1.5 ml (Fisher Scientific, cat. no. 05-408-129) ! CRITICAL Autoclave the tubes before use.
  • Sterile plastic pipettes (Corning, cat. no. 356543 (5 ml), 356551 (10 ml), 356525 (25 ml), and 356550 (50 ml))
  • Sterile tips (MBP ART, cat. no. 2779-HR (P1000), 2769-HR (P200), and 2749-HR (P20))
  • Spray bottle (VWR, cat. no. 23609-182)
  • Air-jacketed 37 °C and 5% CO2 incubator (VWR, cat. no. 10810-902)
  • Biological safety cabinet (Labconco, cat. no. 97000-862)
  • Water bath (VWR, cat. no. 89501-460)
  • Culture microscope (Olympus, cat. no. CKX41)
  • EVOS FL Cell Imaging System (Life Technologies, model no. AMF4300)
  • Keyence fluorescence microscope (Keyence, model BZ-X710)
  • Confocal microscope (Leica, model no. TCS SP8)
  • Stainless-steel surgical blade, sterile, no. 10 (Medicon, cat. no. 01.22.10)
  • Sterile cell strainer (Corning, cat. no. 352340)
  • Petri dishes (non–tissue culture treated), 6 cm (Corning, product no. 430589)
  • Hemocytometer (VWR, cat. no. 15170-208)
  • Nylon, 0.22-μm nylon syringe filter (Celltreat, cat. no. 229775)
  • Centrifuge, 5702 series (Eppendorf, cat. no. 022628102)
  • Falcon tubes (50 ml; Fisher, cat. no. 14-432-22)
  • 5% CO2 tank with plastic hosing leading to culture hood (VWR, cat. no. 89068)

For assembloid formation only

  • Sterile 1.5-ml microcentrifuge tubes (Thomas, cat. no. 1218A70)
  • Scissors (Fisher, cat. no. 08-951-20) and/or razor blades (Fisher, cat. no. 12-640)

For cryosectioning and immunostaining only

  • Microslides, Superfrost Plus (VWR, cat. no. 48311-703)
  • Tissue Path Disposable Base Molds—15 × 15 × 5 mm (Fisher Scientific, cat. no. 22-363-553)
  • Humidified chamber (we use a homemade chamber made from a slide box and wet tissue paper)
  • Elite PAP pen (Diagnostic BioSystems, cat. no. K039)
  • Cover glasses, rectangular (Fisherbrand, cat. no. 22-266882)
  • Cryostat (Leica, cat. no. CM1860)
  • Sable brushes, no. 1, 1.5 mm width × 9.5 mm length (Ted Pella, cat. no. 11812)

For optical clearing of intact spheroids by iDISCO only

  • 4-ml E-C borosilicate glass screw thread vials with TFE-lined caps (Wheaton, cat. no. 03-343-6C)
  • Rectangular cover glasses (Fisher Scientific, cat. no. 22-266973)

For live imaging of cell migration and calcium dynamics only

  • Environmental chamber for confocal microscope (Okolab (CO2 unit: CO2-O2 UNIT-BL (0-20; 1-95); temp chamber: cage incubator, T unit))
  • Glass-bottom plates (Corning, cat. no. 4580)


Reagent Setup

Reconstitution and storage of growth factor and chemical stock solutions

Resuspend FGF2, EGF, BDNF, and NT3 growth factors in sterile 0.1% BSA and DPBS to the desired stock concentration. Stock solution concentrations are as follows:

  • FGF2 stock solution, 20 μg/ml; use at 10 ng/ml for hPSCs (1:2,000) and 20 ng/ml for neural differentiation (1:1,000).

  • EGF, BDNF and NT3 stock solutions, 20 μg/ml; use at 20 ng/ml (1:1,000).

  • Resuspend Amount25 mg of DM in Amount12.52 mL of sterile dimethyl sulfoxide (DMSO) to obtain a Concentration5 millimolar (mM) stock solution; use at a final concentration of Concentration5 micromolar (µM) (1:1,000).

  • Resuspend Amount10 mg of SB in Amount2.60 mL of 100% (vol/vol) ethanol to obtain a Concentration10 millimolar (mM) stock solution; use at a final concentration of Concentration10 micromolar (µM) (1:1,000).

  • Resuspend Amount10 mg of IWP-2 in Amount4.29 mL of sterile DMSO to obtain a Concentration5 millimolar (mM) stock solution; use at a final concentration of Concentration5 micromolar (µM) (1:1,000).

  • Resuspend Amount5 mg of SAG in Amount94.96 mL of sterile DMSO to obtain a Concentration100 micromolar (µM) stock solution; use at a final concentration of Concentration100 nanomolar (nM) (1:1,000).

  • Resuspend Amount5 mg of AlloP in Amount156.99 mL of sterile DMSO to obtain a Concentration100 micromolar (µM) stock solution; use at a final concentration of Concentration100 nanomolar (nM) (1:1,000).

  • Resuspend Amount50 mg of RA in Amount166.42 mL of sterile DMSO to obtain a Concentration1 millimolar (mM) stock solution; further dilute the stock solution to obtain a Concentration100 micromolar (µM) ; use at a final concentration of Concentration100 nanomolar (nM) (1:1,000).

  • Prepare aliquots of stock solutions and store at –80 °C for up to 1 year. Once thawed, stocks can be kept at 4 °C for up to 2 weeks.

Media Preperation

Various media are required; compositions are as indicated below for each specific medium. When ready for use, add aliquots of required media to 50-ml sterile tubes and prewarm in a water bath at 37 °C for <20 min. Avoid cycles of refrigeration and warming.
! CRITICAL Add growth factors and small molecules immediately before use and only after prewarming aliquoted media.

MEF medium (for use in Steps 1 and 2). Prepare the medium as detailed in the table above. This medium can be stored for 1–2 weeks at 4 °C.
hPSC medium (for use in Steps 5–16). Prepare the medium as detailed in the table above. This medium can be stored for 1–2 weeks at 4 °C in the absence of growth factors. Add growth factor (FGF2) just before use.
Neural induction medium (for use in Steps 17–20, on days 0–5 of differentiation). Prepare the medium as detailed in the table above. This medium can be stored for 1 week at 4 °C in the absence of growth factors. Add growth factors (DM, SB, and IWP-2) just before use.


Neural differentiation medium (NM; for use in Steps 21 to 26, on day 6 and onward of differentiation). Prepare the medium as detailed in the table above. The stock can be stored for up to 1 week at 4 °C; add growth factors just before use if required for the culture.

Enzyme stock solution (required for dissociation of cells into a single-cell suspension)

Prepare a stock solution as detailed in the table above. The stock can be stored for 2–3 months at 4 °C.

Inhibitor stock solution (required for dissociation of cells into a single-cell suspension)

Prepare a stock solution as detailed in the table above. The stock can be stored for 2–3 months at 4 °C.

Dispase solution

  • Reconstitute dispase in hPSC medium (without growth factors) to a stock concentration of 1.75 mg/ml. Prepare aliquots of the stock solution and store at –20 °C for up to 1 year.
  • For hPSC passaging, thaw the dispase and dilute in hPSC medium to a concentration of 0.875 mg/ml.
  • For lifting hPSC colonies for sphere formation, dilute the stock solution to 0.35 mg/ml in hPSC medium and warm in a water bath at 37 °C for no more than 10 min.
  • The working dispase solution can be kept at 4 °C for up to 1 week.

Low-Ovo solution (10×; required only for single-cell dissociation)

  • Add Amount3 g of BSA to Amount150 mL of DPBS and mix well.
  • Add Amount3 g of trypsin inhibitor and mix to dissolve.
  • Adjust the pH to 7.4; this requires the addition of ~Amount1 mL of 1 N NaOH.
  • When completely dissolved, bring to a final volume of Amount200 mL with DPBS and filter through a 0.22-µm filter.
  • Prepare 1.0-ml aliquots and store at –20 ˚C for up to 6 months.

High-Ovo solution (10×; required only for single-cell dissociation)

  • AddAmount6 g of BSA to Amount150 mL of DPBS. Add Amount6 g of trypsin inhibitor and mix to dissolve.
  • Adjust the pH to 7.4; this requires the addition of at least Amount1.5 mL of 1 N NaOH. If necessary, add NaOH until the solution is no longer too acidic.
  • Bring to a final volume of Amount200 mL with DPBS. When completely dissolved, filter through a 0.22-µm filter.
  • Prepare 1.0-ml aliquots and store at –20 ˚C for up to 6 months.

Paraformaldehyde

  • Prepare 4% (vol/vol) paraformaldehyde (PFA) by mixing Amount10 mL of 16% (vol/vol) PFA with Amount30 mL of deionized (DI) water, and store at 4 °C for up to 1 week. ! CAUTION PFA must be handled in a safety cabinet and should be disposed of according to institutional guidelines.

Embedding solution

  • Prepare a 30% (wt/vol) sucrose solution by mixing Amount30 g of sucrose with DPBS to a final volume of Amount100 mL .
  • Store the 30% (wt/vol) sucrose solution at 4 °C for up to 2 weeks.
  • Add Amount5 mL of OCT to Amount5 mL of 30% (wt/vol) sucrose to make a 1:1 embedding solution.
  • Shake the solution vigorously until the OCT and sucrose have mixed together.
  • Leave the mixed solution overnight at 4 °C to allow bubbles formed during the mixing process to disappear.

Blocking solution for immunostaining

  • Prepare a blocking solution for immunostaining by mixing 10% (vol/vol) normal donkey serum with 0.3% (vol/vol) Triton X-100 in DPBS.
  • Keep this solution at 4 °C for up to 1 week.


Equipment setup

CO2 tank

  • The 5% CO2 tank setup should have plastic hosing leading to the culture hood and can include multiple three-way stops to split the hosing into multiple lines.
  • To bubble CO2 through solutions, attach a syringe filter to the end of the hosing, and then attach the broken end of a 2-ml pipette and place the tip into the liquid.
  • During dissociation, place a syringe filter on the end of the plastic tubing and set the filter tip into a small hole cut into the lid of the Petri dish (without contacting the solution) so that CO2 can be blown over the digesting solution.
  • If this setup is not available, solutions can also be equilibrated in the incubator.

Troubleshooting
Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.

Ethyl ethanol is flammable and should be maintained in a flame-protective cabinet.

PFA must be handled in a safety cabinet and should be disposed of according to institutional guidelines.
Before start
See "Materials" for reagent setup.
MEF feeder seeding and initiation of hPSC cultures
Coat 10-cm cell culture dishes with 0.1% gelatin for at least Duration01:00:00 at TemperatureRoom temperature .
2h
Thaw one vial of MEFs (containing ~5 × 106 cells) by placing it in a water bath at Temperature37 °C until it is 80% thawed (and for no longer than Duration00:02:00 ).


Briefly spin the cells at Centrifigation200 x g for Duration00:04:00 at TemperatureRoom temperature and plate at a density of 1–2 × 106 cells per 10-cm plate.



The next day, assess feeder quality before use; this will determine the quality of the hPSCs.
Once the MEFs are ready for plating hPSCs (after Duration02:00:00 ), wash the culture plates at least once with fresh DPBS.
Passaging hPSCs on the MEF feeder layer
Remove hPSC medium from the refrigerator, aliquot the required amount into a centrifuge tube (~Amount12 mL per 10-cm plate), wrap the container’s cap with Parafilm to prevent contamination when putting it into the water bath, and warm in a water bath for up to Duration00:20:00 .


Remove the hPSC plate from the incubator and look at it under a microscope.
While under the microscope, mark the differentiated areas on the bottom of the plate, using a marker.
Note
Differentiated colonies may appear to have a hole or a pit in the center of the colony or a border of differentiated cells. (See Fig. 3a of the original publication).

After wiping with a paper towel sprayed with 70% (vol/vol) ethyl alcohol, transfer the plates to the cell culture hood.
Use a pipette tip to carefully remove demarcated differentiated colonies from the plate.
Aspirate the medium and add Amount5 mL of dispase (0.875 mg/ml in hPSC medium) to each culture plate, and place in the cell culture incubator for Duration00:07:00 or until hPSC colonies have lifted.
Transfer the suspended cells from a single plate to a 50-ml centrifuge tube. Use Amount5 mL of hPSC medium with 10 ng/ml FGF2 and 10 µM Y-27632 to wash each 10-cm plate.
Perform this step three times, adding the medium to the appropriate 50-ml centrifuge tube after each wash.
Gently swirl the 50-ml centrifuge tubes to suspend the harvested cells in Amount20 mL of hPSC medium.
Transfer the hPSC medium and suspended cells to a freshly prepared feeder plate (from Step 4).
Gently swirl the plate and slide the plate in a back-and-forth motion on the shelf of the incubator to evenly disperse the cells.
Incubate the cells in the cell culture incubator. Leave the cells undisturbed for the first day after passaging, and replace the medium every day thereafter for 5–7 d.
Note
Critical Step: Replace hPSC culture medium at approximately the same time each day.

To replace the medium, warm aliquoted fresh hPSC medium in a water bath for up to Duration00:10:00 at Temperature37 °C , aspirate old medium from the plates, and add Amount12 mL of fresh medium per 10-cm culture plate.



Monitor the hPSC colonies to determine when they are ready for spheroid formation. This will usually be ~6–7 d from the last passage (depending on the hPSC line); colonies should measure ~1.5 mm in diameter.
Note
See Fig. 3a in original publication for examples of hPSC colonies of appropriate, and inappropriate, morphology.

When cultures are ready, proceed to the next step. Alternatively, continue to passage hPSCs every 5–7 d (depending on the growth rate of each line) by repeating Steps 1–13; however, bear in mind that, ideally, hPSCs should not be kept beyond passage 40.
Note
Critical Step: It is essential for the colonies to be large in order to generate spheroids (i.e., just before the time at which colonies would be passaged, or 1–2 d later).

  • If the colonies are too small, allow more time for growth before subsequent steps.
  • Avoid using hPSC colonies that touch each other because they will detach as large clusters together with the MEF layer.
  • Avoid hPSC colonies that have grown too large and that are differentiating in the center.

Suspending hPSC colonies to form spheroids
Remove the hPSC media from the culture plates. Detach hPSC colonies by incubating them with 0.35 mg/ml dispase (dissolved in hPSC medium; Amount5 mL per plate) for Duration00:30:00 to Duration00:45:00 at Temperature37 °C , 5% CO2.
Note
Check the status of the detachment after Duration00:20:00 and then every Duration00:05:00 thereafter. Different lines will require different durations of dispase treatment. Plates can be gently moved (back and forth) at the end of the incubation period to facilitate the detachment of curled hPSC colonies from the feeders. Almost all feeders should remain attached to the 10-cm plate, whereas the hPSC colonies should come off. Intact colonies should curl up and lift off the plate (see Fig. 3b in original publication).



3h
Gently transfer the floating colonies with a pre-wet 25-ml pipette to a 50-ml Falcon tube.
Once the colonies settle at the bottom of the tube, aspirate the media and gently wash the colonies two times with Amount20 mL of warm hPSC medium (without growth factors).
Note
Critical Step: Do not centrifuge. Let the colonies settle at the bottom of the Falcon tube.

Gently resuspend 30–50 suspended colonies in Amount12 mL of neural induction medium (the medium should also contain 5 µM DM, 10 µM SB, and 10 µM Y-27632) and transfer to a 10-cm ultra-lowattachment plate.
Note
Caution: It is important to distribute the spheroids evenly once the plate is placed in the incubator to avoid clustering and fusion of colonies at the center of the plate.

Denote this day as day 0. Place the plate in the incubator atTemperature37 °C , 5% CO2 for Duration48:00:00 . Keep the spheroids in the incubator on day 1 without any medium change to allow the spheroids a day to recover.

Neural induction
Critical: Minimize the overall time that the spheroids are kept outside of the incubator at all stages of differentiation.

(Day 2)
After Duration48:00:00 of incubation, i.e., on day 2, replace the medium with Amount12 mL of neural induction medium (with DM and SB but without Y-27632).

To change the medium, gently transfer the spheroids to a 50-ml Falcon tube and carefully aspirate the medium once they settle. Incubate the cells for a furtherDuration24:00:00 .

(Day 3)
Replace the medium as described in Step 18 and incubate the cells for a further Duration24:00:00 .
(Day 4)

If you plan to differentiate hSSs (when you reach Step 22, Step 24), replace the medium as described in Step 18, but include Concentration5 micromolar (µM) IWP-2 in the medium in addition.

If you plan to generate hCSs, replace the medium as before.

Incubate the cells for a further Duration24:00:00 .
Note
Critical Step: IWP-2 should not be thawed at RT, as it will precipitate. Frozen aliquots should be placed directly into a water bath before use.

Patterning and differentiation
From day 6 onward, replace the medium daily with NM in place of the neural induction medium. Use NM supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF. In addition, include 5 µm IWP-2 in the medium if you are generating hSSs.
Change the medium daily on days 6–11. At day 6, neural spheroids should be ~0.3–0.5 mm in diameter (Fig. 3c of original publication).
Note
The medium can be changed as described in Step 18 or by gently tilting the plate (allowing the spheroids to settle) and removing the remaining medium.

From day 12 onward, the medium requirements for the generation of hCSs and hSSs deviate further.

For generation of hCSs go to step 23.

For generation of hSSs go to step 24.

For generation of hSS-ISRAs go to step 25.
Note
After neural spheroids grow larger (>2–3 mm in diameter), no more than 30 spheroids should be maintained per 10-cm plate.

Critical Step: Neural spheroids tend to fuse to each other as they get larger (Fig. 3d of original publication). When changing the medium, be sure to separate them by either pipetting up and down gently with a 10-ml pipette or by using a P10 pipette tip to separate them when they are bigger. If spheroids fuse during later time points (after ~day 50), it may greatly compromise the outcome.

Generation of hCSs
From days 12 to 15, continue to replace medium daily with NM supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF.
From day 16 onward, change the medium every other day until day 25. Continue to use NM supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF.
From days 25 to 43, replace FGF2 and EGF with 20 ng/ml BDNF and 20 ng/ml NT3.

Perform medium changes with Amount14 mL - Amount15 mL of supplemented NM per 10-cm plate every 2–3 d.

Go to step 27

Generation of hSSs
From days 12 to 23, supplement the NM (already containing 20 ng/ml FGF2, 20 ng/ml EGF, and 5 µm IWP-2) with Concentration100 nanomolar (nM) SAG.

Perform medium changes with Amount14 mL - Amount15 mL of supplemented NM per 10-cm plate every day.
Note
Critical Step: Note that early hSSs can be smaller in diameter in comparison with early hCSs during 12–20 d of differentiation

On day 24, replace medium with NM without additional growth factors.
From days 25 to 43, replace FGF2 and EGF with 20 ng/ml BDNF and 20 ng/ml NT3.

Perform medium changes with Amount14 mL - Amount15 mL of supplemented NM per 10-cm plate every 2–3 d.

Go to Step 26.
Generation of hSS-ISRAs
On days 12, 13, and 14, supplement the NM (with IWP-2) with 100 nM SAG.

Perform medium changes with Amount14 mL - Amount15 mL of supplemented NM per 10-cm plate every day.
From days 12 to 15, add Concentration100 nanomolar (nM) RA (in addition to the IWP-2) to the neural induction medium (Fig. 2c of original publication).
Note
Caution: RA is air- and light-sensitive. Minimize light exposure.

From days 15 to 23, add Concentration100 nanomolar (nM) AlloP (in addition to the IWP-2) to the neural induction medium (Fig. 2c of original publication).
On day 24, replace medium with NM without additional growth factors.
From days 25 to 43, replace FGF2 and EGF with 20 ng/ml BDNF and 20 ng/ml NT3.

Perform medium changes with Amount14 mL - Amount15 mL of supplemented NM per 10-cm plate every 2–3 d.

From day 43 onward, perform media changes with Amount17 mL - Amount18 mL of NM without growth factors every 4 d. Using careful sterile technique, spheroids can be maintained for many months in culture, if desired (Fig. 3d of original publication).

Carry out any additional procedures at time points determined by your experiment.

Examples of additional procedures that can be carried out on the spheroids include:
Step case

Generation of forebrain assembloids w/ hCSs + hSSs
4 steps

For assembly of forebrain spheroids, transfer one hCS and one hSS to a 1.5-ml microcentrifuge tube that is resting in a standard microtube rack (Fig. 4f of original publication).

Denote the day of assembly as day 0. The size of the spheroids to be assembled should not exceed 3 mm, as they are challenging to fuse once they are too large.
Note
Critical Step: Once in the tube, make sure that there are no bubbles underneath the spheroids and that they rest next to each other in contact at the bottom of the tube and are not stacked up vertically.

Incubate the spheroids for at least Duration42:00:00 in Amount1 mL of NM, completely replacing the medium on day 2.
Note
Caution: Because the assembly interface is fragile at this stage, perform the medium change in the tube very carefully.



Check that fusion is complete.

Fusion is considered complete when the two spheroids are inseparable with gentle shaking of the Eppendorf tube (Fig. 4g of original publication).
After fusion, use a P1000 pipette with the tip cut off for a larger bore opening to transfer the assembled hCS–hSSs back to the ultra-low-attachment 10-cm plates. If fusion is carried out between days 60 and 90, when this process is most efficient, hCS–hSS assembly is usually complete after 3–4 d.
Note
Earlier time points are also permissive. Assembly of hCS–hSSs at later stages of differentiation (after day 90) may take longer (up to 7 d).