Highly reproducible human brain organoids recapitulate cerebral cortex cellular diversity


 Human brain organoids hold an unprecedented opportunity to observe, perturb, and study the early stages of human cortical development. Several protocols to generate brain organoids have been described in recent years[1, 2]. However, incomplete characterization and lack of organoid-to-organoid reproducibility has limited their application as an experimental model[3]. Here we describe a detailed protocol for the generation of human dorsal forebrain organoids that show highly reproducible generation of the rich diversity of cell types present in the developing human cerebral cortex. This protocol is a modification of a previous method described by Kadoshima et al.[4]. We also include a detailed description of the protocol used to dissociate organoids into single cells for single-cell RNA-sequencing.

Store at -20 °C for up to 1 year. Avoid multiple freeze-thaw cycles.
Store at -20 °C for up to 1 year. Avoid multiple freeze-thaw cycles.
Store at -20 °C for up to 1 year. Avoid multiple freeze-thaw cycles.

1.4) Heparin
The concentration used here is measured in mg/ml, however the manufacturer reports quantity in units. Generation of reproducible human brain organoids

Maintenance of human pluripotent stem cells (hPSCs)
Maintain feeder-free hPSCs on TC-treated cell culture dishes pre-coated with Geltrex, in mTESR1 medium with 100 U/mL penicillin and 100 μg/mL streptomycin, in a humidi ed incubator at 37˚C and 5% CO 2 .
Detailed protocol for thawing, maintenance, and passaging of hPSCs can be found in Arlotta et al., 2017 [5] . · CRITICAL STEP: It is recommended to routinely con rm karyotype stability (suggested: every 5 passages) and make sure that stem cells are mycoplasma-free (on a weekly basis). MycoAlert PLUS Mycoplasma Detection Kit can be used to detect mycoplasma contamination. Single-cell dissociation should be avoided during passaging. The use of non-enzymatic methods to detach cell clumps, for instance Gentle Cell Dissociation Reagent, is recommended.
Day 0 -Cell aggregate formation in CDM I Media 1. Start with hPSCs that are 80-90% con uent. · CRITICAL STEP: For the generation of brain organoids, it is recommended to use healthy viable hPSCs (mycoplasma-free, karyotypically normal and below passage 50) with typical morphological features of pluripotent cells (tightly packed colonies of round cells with large nuclei and nucleoli), with no sign of differentiation. Optimal stem cell culture practice and attention to details are essential requirements for the formation of healthy dorsal forebrain organoids.
2. Before starting cell dissociation, prepare the following mix (Seeding Medium): -CDM I (10 mL) -ROCK inhibitor [6]  4. Add Accutase to the dish and incubate for approximately 4-5 minutes at 37˚C. · CRITICAL STEP: Different incubation times may be required depending on cell con uence or for different hPSCs. 5. Use a 1 mL pipette to dissociate the cells. · CRITICAL STEP: Avoid rough pipetting to prevent damage to cells, which can lead to cell death. 6. Add mTESR1 to dilute the Accutase suspension containing the dissociated cells and transfer content to a 15 mL conical tube.
7. Centrifuge the cells. 8. Aspirate the supernatant and resuspend the cells in CDM I. 9. Centrifuge the cells. 10. Aspirate the supernatant and resuspend the cells in 1mL of CDM I. 11. Count the cells.

Dilute the proper volume of cell suspension (containing 900,000 cells) from
Step 10 into 10 mL of Seeding Medium.
14. Keep the plate in a humidi ed tissue culture incubator at 37 °C and 5% CO 2 .
Day 1 -Observe the 96 multiwell plate under the microscope to con rm aggregate formation. · CRITICAL STEP: Aggregates with de ned and homogeneous borders should be visible, although a few dead cells might appear around the EB (Fig. 1). 17. Using a multichannel pipette, add 100 μL of mix to each well. · CRITICAL STEP: before adding fresh medium, gently agitate the medium inside each well by using a multi-channel pipette. This step helps detach dead cells that might surround the aggregate as a result of the dissociation step.
18. Return the plate to a humidi ed tissue culture incubator at 37 °C and 5% CO 2 . 43. Maintain spinner asks on a magnetic stirrer at 56 rpm inside a humidi ed tissue culture incubator at 37 °C and 5% CO2.
Single-cell dissociation of brain organoids for single-cell RNA Sequencing (10x Genomics Chromium platform) The following protocol is a modi cation of the Worthington Papain Dissociation kit manufacturer's protocol, as used in previous works from our lab [3,8] .
17. Dilute and count the cells.

Troubleshooting
• Carefully observe cell aggregates under a microscope, throughout the differentiation process. Take pictures of the same cell aggregates (at least three different cell aggregates) every 2-3 days to monitor morphological changes (Fig. 1).
• Validate each experimental batch by performing IHC for the neuronal marker MAP2 and the dorsal forebrain progenitor markers PAX6 and EMX1. The expression of EMX1 is indicative of successful differentiation. Staining for activated Caspase 3 should also be included to assess organoid health. Perform the IHC analysis on at least 3 individual organoids derived from the same batch, at multiple time points. A detailed explanation on the use and working dilutions for the antibodies used for organoid characterization can be found in the associated publication [7] .
• This protocol has been successfully used to generate organoids from several pluripotent stem cell lines, including multiple hiPSC and hESC lines, from both male and female lines [7] . However, we cannot exclude that modi cations to the protocol may be required to increase the e ciency of neural differentiation of speci c cell lines.

Anticipated Results
In general, when healthy (karyotipically normal, mycoplasma-free) viable hPSCs, 80-90% con uent, showing good pluripotent morphology (no signs of differentiation) are used, the e ciency of forebrain cell type generation by using this protocol is ~90-95% [7] .