Generation of human midbrain organoids from induced pluripotent stem cells

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.


Background
Parkinson's disease (PD) is a neurodegenerative disorder, affecting more than 1% of the population over 65 years of age. The majority of cases are idiopathic, while about 10% have been linked to genetic mutations. Classical hallmarks of PD are the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta, accompanied by the presence of neuronal inclusions called "Lewy bodies". Several cellular pathways have been implicated in PD pathogenesis, including mitochondrial dysfunction 1-3 , perturbed neuronal activity 4,5 and dysregulated protein homeostasis due to lysosomal, autophagy and proteasomal defects 6-9 . However, there is no treatment to halt the progression of the disease. To date, treatment of PD is limited to symptom management. It is therefore necessary to refine the models we use in fundamental research to understand the pathophysiology of PD and to develop more effective therapeutic strategies.
In 2006, Drs. Kazutoshi Takahashi and Shinya Yamanaka described the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) 10 . Since their discovery, this technology has opened up many new avenues of investigation, including research for PD. With their self-renewal abilities and potential to be differentiated into disease-relevant cells from all three developmental lineages, iPSCs provide a unique tool to study PD within a human neuron, without the difficulties in obtaining neurons from a human brain 11 . iPSCs can be directly reprogrammed from skin, blood or urine of an individual without raising the ethical concerns previously triggered by the use of fetal stem cells. In 2009, Soldner et al. were the first to describe the generation of an iPSC cell-line from a patient with sporadic PD, and the subsequent differentiation of these cells into dopaminergic (DA) neurons 12 . Taking advantage of iPSC technology, many studies have started to investigate the pathological mechanisms of PD in iPSC-derived DA neurons from patients, compared to neurons from healthy individuals  .
Recently, the development of organoids has become a major technological advance in the stem cell field and represents a bridge between traditional 2D cultures and in vivo mouse models. In 2013, Lancaster et al. described a novel 3D model called a cerebral organoid, that recapitulated different areas of the human brain 34 . Kept in culture, these organoids formed a complex self-organized neuronal tissue composed of a mixed population of neurons, astrocytes and oligodendrocytes. In contrast to neurospheres, the cells were organized in layers that, at early stages, included a ventricular zone composed of progenitors. Neurons within these organoids were functional and had spontaneous electrical activity in networks. Interestingly, brain organoids could be cultured for long periods to obtain morphologically and functionally mature cells, in contrast to neurosphere cultures [34][35][36][37] .
Since 2013, different types of brain organoids have been generated based on adaptations of the initial protocol published by Lancaster. Earlier, the protocols involved no external addition of growth factors in the medium, resulting in self-differentiating cells. However, different laboratories now directly drive the stem cells towards specific fates. The key to efficient brain organoid generation is a defined combination of inductive signals and physical factors that drive pluripotent stem cells to form 3D brain organoids. The modulation of these factors gives rise to multiple types of brain organoids that can be used to model neurodevelopmental and neurodegenerative disorders that affect distinct regions within the brain. Protocols now exist for making human cerebral 34,38,39 , forebrain-like (dorsal and ventral) 40,41 , cerebellar 42 , cortical-like (dorsal and ventral) 43,44 , hippocampal and choroid plexus-like tissue 45 , midbrain 35,36,41 , hypothalamic 41 , pallium and subpallium 46 brain organoids.
iPSC-derived human brain organoids recapitulate brain development and can be used to study normal neurodevelopment. Brain organoid development recapitulates the early to mid-fetal development, and the epigenomic signatures of the human foetal brain 41,47,48 . So far, cerebral organoids have been used to study pathologies including microcephaly 34 , Zika virus infection [49][50][51][52] , and autism spectrum disorders 46,53 .
Recently, human brain organoids have been used to investigate aspects of neurodegenerative disorders. Two groups generated cerebral organoids from iPSCs of Alzheimer's disease (AD) patients carrying familial mutations for presenilin 1 or amyloid precursor protein duplication, and successfully recapitulated the aggregation of beta-amyloid protein and tau pathology (hyperphosphorylation and aggregation), the two neuropathological markers of AD, in a human model. Treatment of the 3D cultures with drugs targeting either amyloid-beta aggregation or tau phosphorylation decreased the pathological markers 54,55 . These promising results demonstrated that human brain organoids represent a relevant model for drug discovery. The development of different types of brain organoids represents a major advance in the stem cell field. In particular, the development of midbrain organoids represents a new drug discovery tool for PD. Two groups published similar protocols to generate the human midbrain organoids, based on specific inductive signals introduced at specific stages in the 3D cultures to drive the stem cells towards a midbrain fate 35,36 . They showed that the midbrain organoids are composed of functional midbrain neurons producing neuromelanin granules, a by-product of dopamine synthesis. Of the neuronal population, 30% was myelinated due to the presence of oligodendrocytes. Interestingly, Monzel et al., showed the presence of nodes of Ranvier and spontaneous saltatory transmission 35,36 . Considering the mix of neuronal populations connected within the midbrain organoids, they represent an interesting model to discover new pathological mechanisms involved in PD.
In this paper, we provide a standardized protocol for a robust derivation of iPSCs lines into 3D midbrain organoids. This protocol is an adaptation of the Nature protocol paper initially published by Lancaster et al. in combination with discoveries from Jo et al. and Monzel et al. [34][35][36] in order to successfully produce high quality midbrain organoids. We also describe a cryosectioning protocol that we adapted to produce high-quality histological sections from midbrain organoids, overcoming difficulties resulting from the particular texture of cultured tissue as well as their small size, relative to rodent brains. Taken together, this article extensively explains the methods involved in generating these iPSC-derived organoids and their histological analysis.

Materials
The materials, reagents and equipment listed in this document can be substituted for comparable items. However, the performance of the protocol may not be the same and may need to be optimized or redeveloped upon significant modifications to the materials and/or methods. It is also important to note that there is a significant lot-to-lot variability for certain reagents. In order to monitor this variability, we recommend a systematic test of new batches.
List of materials, reagents and equipment for 3D midbrain generation For growing human iPSCs, the quality of reagents is critical. Variability in the quality of any of these materials or in associated manufacturing processes will lead to inconsistent quality, which has been reported to negatively impact human iPSCs cultures. See Table 1 and Table 2.

Background information on media
Neuronal induction medium: A cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) enhances survival of iPSCs when dissociated to single cells and improves embryoid bodies formation 56 . Midbrain is of ectodermal origin, thus neuroectodermal differentiation towards a floor plate can be induced with dual-SMAD inhibition factors, Noggin and SB431542 and a Wnt pathway activator, CHIR99021 (chemical name: Complementary to these factors, heparin plays a role in enhancing the activity of Wnt signaling 58 . 2-mercaptoethanol regulates oxidative stress to maintain cell growth and avoid cell death due to toxicity. Retinoic acid, a metabolite of vitamin A, is a potent caudalizing factor that we exclude from our media to promote midbrain differentiation. See Table 3.

Midbrain patterning medium:
The patterning can be influenced by sonic hedgehog, SHH and fibroblast growth factor FGF8 as they are responsible for guiding the cells towards the mesencephalic fate 59 . To avoid the dorsal influence on organoids patterning, B27 supplement without vitamin A is considered appropriate 60 . See Table 4.

Tissue Induction Medium:
The presence of insulin and laminin promote the growth of tissue embedded. See Table 5.

Final Differentiation Medium:
The presence of brainderived neurotrophic factor (BDNF), is reported to play a potential role in developing cholinergic, dopaminergic, serotonergic and gamma-aminobutyric acid (GABA) ergic neurons, along with promoting the function and survival of other neuronal populations 61 . The other growth factor, glial cell-derived neurotrophic factor (GDNF) also affects neuronal differentiation, maturation and neurite growth by enhancing myelination. It has also been reported to induce a dopaminergic phenotype 62 . See Table 6.
iPSC lines For growing midbrain organoids from human iPSCs, the quality of iPSCs is critical. Variability in the iPSCs maintenance will negatively impact midbrain organoids generation (section "Protocol description for iPSC culture and maintenance"). The observations provided in this method were generated with at least 6 independent batches derived from two iPSC lines from healthy individuals (NCRM-1 and XCl-1) or an iPSC line from a patient with PD (EDI001A named as SNCA_Tri in Figures) ( Table 7).
List of material, reagents and equipment for 3D midbrain histological processing and cryosections See Table 8. See Table 9.

Method
Protocol description for iPSC culture and maintenance General principles for culturing and maintaining human iPSCs. Human iPSCs are sensitive to many stresses, including shear stress, heat shock, and changes in media formulation and must be manipulated with extreme care at all steps of the protocol.

Technical and safety considerations for manipulating IPSCs
• The iPSC colonies should not have been passaged more than 10 times after thawing.
• The cells need to be a minimum of passage 2 after thawing for generation of midbrain organoids.
• Do not work with colonies that present with differentiated areas (Figure 1a, Figure S1a). We recommend removing the differentiated areas during daily maintenance and to never exceed 5% of differentiated areas prior to organoid generation for optimal organoid formation.     • Sterile technique must be used at all times when working with cells or in preparing reagents and materials.
• Human iPSC lines are to be handled within a Class II biosafety laminar flow hood to protect the worker from possible biohazardous agents.   • Prepare DMEM/F-12 + GlutaMAX TM -I solution by removing 5 mL of DMEM/F-12 + GlutaMAX TM -I from a 500 mL bottle and adding 5 mL of Antibiotic-Antimycotic.
Place the bottle at 4°C until cold.
• Once the Matrigel® is thawed and DMEM/F-12 + Gluta MAX TM -I is cold, prepare a coating solution diluted as per manufacturer instructions and mix well.
• Immediately use the diluted Matrigel® solution to evenly coat the tissue culture dishes (2 ml/ 60 mm dish and 5 ml/ 100mm dish) by swirling the tissue culture dish in each direction, multiple times. Incubate at 37°C for a minimum of one hour before use.
• Meanwhile, warm up mTeSR TM 1 and DMEM/F-12 + GlutaMAX TM -I medium at room temperature (RT) for a minimum of one hour. Do not use a water bath during this process because the media components would be degraded.
• Once the tissue culture dish is coated and mTeSR TM 1 and DMEM/F-12 + GlutaMAX TM -I medium are warmed, get the frozen cryovial of iPSCs from the liquid nitrogen storage container, and quickly thaw the vial by warming in a 37°C water bath. Continuously shake the cryovial until only a small frozen pellet remains.
• Using a 5 mL glass pipette, transfer the contents of the cryovial to a 5 mL solution of DMEM/F-12 + GlutaMAX TM -I (with Antibiotic-Antimycotic), in dropwise manner and gently pipette up and down 1-2 times.
• Centrifuge the cells at 1200 rpm for 3 minutes at RT. After centrifugation, aspirate the medium, leaving the cell pellet intact. Using a 5 mL glass pipette, gently resuspend (1-2 times) the cell pellet in 3 mL of mTeSR TM 1 medium containing Y27632 (1:1000 dilution).
• Take the coated tissue culture dish from 37°C and aspirate out the coating medium. Transfer the cells in mTeSR TM 1 medium to the coated dish and place in a 37°C, 5% CO 2 incubator. Do not disturb the dish for 24h, to allow the cells to attach.
• Change the medium every day with mTeSR TM 1 without Y27632, until the cell confluency reaches 70% and cells are close in contact.
• Daily, visually identify regions of differentiation under the microscope and remove them by aspiration under the sterile hood ( Figure S1a).

Passaging of human iPSC lines
• When the cells reach 60-70% confluency, they are ready to be split.
• Aspirate out the medium from the dish and wash the cells with 3 mL DMEM/F-12 + GlutaMAX TM -I (with Antibiotic-Antimycotic).
• Add gentle cell dissociation medium (5 ml for 100 mm dish and 1 mL for 60 mm dish) at RT until the cells at the edge of the colony begin to detach from each other. (Note: Do not leave cells for longer as the cell viability will be affected) • Aspirate out the gentle cell dissociation medium from the dish and wash the dish with DMEM/F-12 + GlutaMAX TM -I.
• Add 5mL DMEM/F-12 + GlutaMAX TM -I to the dish and gently detach the colonies by scrapping with a cell scraper. Using a glass pipette, transfer the detached cell aggregates in a Falcon tube.
• Centrifuge the Falcon tube with cells at 1200 rpm for 3 minutes.
• After the centrifugation is done, check the pellet and aspirate out the supernatant (do not let the pellet dry so leave 100 µL-200 µl DMEM/F-12 + GlutaMAX TM -I after aspiration).
• Using a glass pipette, resuspend the pellet gently to break the cell aggregates in mTeSR TM 1 medium with 10µM Y27632.
• For seeding cells, add 10% of the original cell suspension to a new coated dish when passaging between similar size dishes. For a small to large dish passage-take 20% of the original cell suspension and from a large to small dish, take 5%.
• Incubate the dishes at 37°C until the colonies reach 70% confluency.
Freezing of human iPSC lines • As described in the previous section, detach cells with gentle cell dissociation medium, and determine cell number with a cell counter prior to pellet the cells by centrifugation.
• Gently aspirate supernatant. Gently resuspend the cell pellet in FBS in half the volume needed to result in 1 mL of cell aggregate mixture per cryovial containing 1.5-2 millions of cells.
• Add an equal volume of 20% DMSO/FBS to the cell aggregate mixture to obtain a final DMSO concentration of 10%. Mix well with three up and down.
• Transfer 1 mL of cell aggregate mixture to each cryovial.
• Place cryovials in a cryo-box and transfer cryo-box to -80°C for overnight storage.
• The following day, transfer cryovials to liquid nitrogen.

Generating midbrain organoids
Eight days are necessary to generate midbrain organoids from iPSC colonies. Then, the organoids are grown with constant agitation for maturation. The timeline is described in Figure 1b.

Seeding of iPSCs -Day 0
• Start with one 10 cm dish of iPSCs at 70% confluency, cultured in mTeSR TM 1 to obtain 96 midbrain organoids from a 96-well ultra-low attachment plate.
• Dissociate cells using Accutase by removing medium from cells, wash cells once with DMEM/F-12 + GlutaMAX TM -I + Antibiotic-Antimycotic and add 5 ml Accutase at RT. Incubate cells for 3 minutes at 37°C, then stop reaction with DMEM/F-12 + GlutaMAX TM -I + Antibiotic-Antimycotic. Transfer the medium with the cells into a 15 mL Falcon tube and centrifuge for 3 min, at 1200 rpm in a regular Falcon centrifuge. Remove the supernatant.
• Using a 1mL tip combined with a 200 µl tip resuspend cells in 5 ml of neuronal induction medium (Table 3) by pipetting gently up and down 3 times. Count cells. Note: The 200 µl tip is at the end of the 1 ml tip.
• Plate 10,000 cells/well in an ultra-low attachment 96 well U-bottomed plate with a multichannel pipette and add neuronal induction medium (Table 3) to a total of 200 µl/well with a multichannel pipette.
• Centrifuge the plate for 10 min at 1200 rpm at 37°C.
• Incubate cells for 48 hrs at 37°C in a regular cell incubator.

Change medium -Day 2 Observation: EBs should have smooth and round edges (Figure 1c)
• Change media to neuronal induction medium WITHOUT ROCK inhibitor using a multichannel pipette.
• Incubate cells for 48 hrs at 37°C in a regular cell incubator.

Change medium -Day 4
Observation: EBs should reach a diameter of >300 µm (typically 400-600 µm) with smooth and round edges • Change medium to midbrain patterning medium (Table 4) using a multichannel pipette.
• Incubate cells for 48 hrs at 37°C in regular cell incubator.

Change medium and embedding in Matrigel® -Day 7
Observation: Neuroectoderm buds should have started to extrude before the embedding (Figure 1d) • Transfer aliquots of Matrigel® reduced growth factors to ice and let it reach 4°C.
• Remove the medium from each well with a multichannel pipette and add 30 µl of reduced growth factor Matrigel® with the manual repeater-pipette and sterile distritips.
• Incubate the plate for 30 min at 37°C in a regular cell incubator.
• Add 200 µl of tissue induction medium (Table 5) and incubate for 24 hours at 37°C in a regular cell incubator.
• Autoclave a box of 1mL cut tips if not already done.

Transfer the organoids to final differentiation medium -Day 8
Observations: Neuroepithelium should be more developed (Figure 1e) • Add 3 mL of final differentiation medium per well of an ULTRA LOW ATTACHMENT 6 well plate.
• Put the 6 well plates on the orbital shaker (set at 70 rpm for the orbital shaker listednote: speed setting would change based on the shaker diameter) in the 37°C regular cell incubator and change the medium every 2-3 days using final differentiation medium (Table 6). When the midbrain organoids reach 1 mm in diameter, increase the final differentiation media volume to 5 mL to provide enough nutrients (Figure 1f).
Note: By putting 5 organoids per well, we limit the number of media changes to three times a week once the hMOs attain the maximum size of 4mm.
Observations: From day 1 in final differentiation media to day 50 the organoids should grow from 600 µm to approximately 4 mm (Figure 2a.b).
Histological processing of organoids by cryosectioning Tissue fixation and cryoprotection • Remove medium from plates and fix organoids with submersion in fresh 4% formaldehyde solution for 1h at RT or O/N at 4°C in fume hood.
HAZARD WARNING: Use care handling formaldehyde solutions. Follow instructions according to the product MSDS.
• Wash organoids with PBS 3 times for 5 min to remove formaldehyde. • Incubate organoids in 20% sucrose solution at 4°C until the organoids sink. This is usually achieved O/N or after 3 days.

NOTE:
Do not extend the incubation for longer than 3 days, as this impacts the quality of sectioning. In rare occasions, the tissue does not sink after a long time because it includes low-density components such as Matrigel®. However, this does not impact subsequent procedures. We recommend removing any trace of Matrigel® prior to sucrose solution immersion.

Block embedding
• Transfer organoids from the sucrose solution to a cryomold using a pipette with a cut tip. We typically embed up to 9 organoids per block. If embedding different types of organoids (different ages, cell lines, etc.) in the same block, care must be taken not to mix the organoids.
• After all organoids are placed in the mold, remove all the sucrose solution with a paper tissue, taking care that the organoids do not stick to the paper.
• Slowly pour optimal cutting temperature (OCT) mounting medium directly on top of the organoids to ensure they stay on the bottom of the cryomold. If embedding different types of organoids, be careful to maintain their organization.
• Use a needle to place organoids in the desired positions, while taking care not to move organoids upwards. Space the organoids about 1 mm from each other.
• Freeze organoids by placing the mold in a -80°C freezer or in the gaseous phase of a liquid N 2 container. When moving the mold, take care not to tilt excessively, which may displace the organoids.
• Once completely frozen, blocks may be stored long term inside a closed container to prevent drying in a -80°C freezer.

Cryosectioning
• Set cryostat temperature and place all blocks to be cut in the same session inside the cryostat chamber to equilibrate the temperature. NOTE: The relationship between the cryostat temperature and the actual temperature at the block surface after mounting varies according to the cryostat model. Using a thermal probe, we found that a surface temperature of -9°C allows easy production of high-quality samples. However, the precise setting necessary to achieve this temperature must be determined for each machine.
• Prior to, or shortly after, removal of the block from mold, cut one corner of the block to keep track of the block orientation in the subsequent steps.
• Trim the block edges using a razor blade, maintaining a margin of 1-2 mm of OCT around the area containing organoids.

WARNING:
Use care when handling the razor blade inside of the cryostat. Avoid manually cutting blocks that are not equilibrated with the cryostat temperature, as these become harder and need more strength to be cut, which may lead to injuries.
• Pour an amount of OCT on the sample holder (chuck) sufficient to cover the entire bottom surface of the block.
• Press the block on top of the OCT layer on the sample holder using a heat extractor to orient the block as horizontally as possible. Wait until OCT freezes completely.
• Place the mounted block in the microtome head and cut sections in the desired thickness. We routinely produce sections with a thickness ranging between 10 and 20 µm.
• If necessary, flatten sections using a pair of brushes and pick sections using a slide kept at room temperature (direct mount method).
• Let slides air dry for 30-60 min and proceed with histological staining. The slides may also be kept in boxes at -80°C for long term storage.
Immunostainings, images acquisition, single cell RNA sequencing and statistical analysis Immunostaining and Fontana Masson staining. Cryosections were rehydrated in PBS for 15 min and surrounded with a hydrophobic barrier using a barrier pen. The sections were then blocked for one hour at room temperature in a humidified chamber, with blocking solution (5% of normal donkey serum, 0.05% BSA, and 0.2%Triton X-100 in PBS. They are then incubated overnight at 4°C with primary antibodies diluted in blocking solution (See Table 9). The following day, cryosections were washed three times in PBS, fifteen minutes each, and then incubated in secondary antibodies diluted in blocking solution (See Table 9) for one hour at room temperature. Then we washed the sections three times in PBS for fifteen minutes each. Hoerscht (diluted 1/5000 in PBS) was incubated 10 min on sections, followed by a wash in PBS for 10 min. Finally, we mounted the section with an aqua-mounting media and visualized the staining under a confocal microscope (Figure 3).
Fontana Masson stainings (Figure 4b) were performed with an Abcam staining kit (#ab150669) following provider's instructions on regular paraffin sections of hMOs.
Imaging. iPSCs colonies (Figure 1a, Figure S1a) were imaged with an inverted microscope Motic AE2000 and the Moticam BTO camera associated, while the EBs images (Figure 1c-f) were taken with a transmitted light microscope EVOS XL Core. The hMOs ( Figure 2 and Figure 4a, Figure S1b) were imaged with a ZEISS Stemi 508 stereomicroscope combined with a ZEISS Axiocam ERc 5s camera. The fluorescence images (Figure 3, Figure S1c) were acquired with a Leica TCS SP8 confocal. Fontana Masson stainings (Figure 4b,d) were acquired with a clinical microscope Olympus BX46 and an Olympus DP27 digital color camera associated. Raw fluorescent images were opened in ImageJ software (version 2.0.0-rc-69/1.52i) with a red, blue, green or yellow color associated to each channel, before all images were merged to create a merged image. Black dots from Fontana Masson stainings pictures were extracted with colorimetric selection from GIMP software (version 2.8.22) and quantified by ImageJ (version 2.0.0-rc-69/1.52i) following the method described in 63. Briefly, using GIMP software the pixels associated with neuromelanin staining were colored extracted, and quantitating the number of extracted pixels using Image J. A histogram of the image was created, which separates the total number of pixels in the image into 255 color categories spanning the visible spectrum. The peak corresponding to the brown-black colour i.e. neuromelanin was determined by cutting and summing the appropriate counts from each channel of the melanin peak. Single Cell RNA sequencing. After dissociation, the single cell suspension in PBS with 1% BSA was put on ice. Cell viability was determined with live dead staining kit and approximately 5000 cells were loaded per lane in 10X Genomics Chromium 2 single cell sequencing chip. The samples were processed following the 10X protocol to prepare cDNA libraries for next-gene sequencing. The sequences were aligned to the human genome (CRCh38) and de-multiplexed to match RNA sequences with cell barcodes using 10Xcell Ranger. The R package Seurat used to analysis the single cell libraries (R notebook appended). The sequences quality was confirmed by checking the number of unique RNA sequences for each cell (nFeature_RNA) and the total number of RNA sequences in each cell (nCount_RNA), where were both in the expected range. The percent of total RNA that was from mitochondrial RNA was calculated, very few cells had over 12% mitochondrial RNA indicating that most cells were alive with intact mitochondria at the time of sequencing. The two hMOs single cell libraries were combined and Louvain nearest neighbor network detection with a resolution of 0.2 was used to cluster cells after Principal Component Analysis for dimensional reduction. The resulting clusters were annotated using a combination of 1) comparing the topmost differentially expressed genes (DGE) distinguishing each cluster and 2) the expression levels of accepted cell type markers. The DGE were determined between cluster X and all other clusters. The gene marker lists for each cell type can be found in the R notebook.
To distinguish between similar clusters the DGE between cluster X and Y were calculated, identifying the markers distinguishing these two clusters. The number of cells expressing TH compared to the total number of cells and the number of cells in each cluster was calculated to get the proportion of TH positive cells.
Statistical analysis. The statistical analysis was performed in GraphPad Prism v7.0. For the quantification of neuromelanin granules, we performed a normality test followed by a parametric unpaired t-test, p***<0.001.

Results and observations
We observed that good quality of iPSCs is a primary determinant in to successfully generating high-quality of hMOs. iPSCs colonies are maintained daily and passaged in order to present no differentiated area (Figure 1a). If the colonies present with less than 5% of differentiated areas after the precautions described, we removed the differentiated areas prior to the generation of hMOs to ensure an optimal quality of hMOs ( Figure S1a). The process to generate hMOs from iPSCs, at 70% confluency, takes eight days (Figure 1b). From this point, embryoid bodies formed from the iPSCs, were differentiated toward midbrain fate in stationary culture and embedded in Matrigel ® to promote the formation of the tissue. Indeed, we observed the progressive appearance of the tissue within the EB (Figure 1c-f). Once the EBs presented with multiple bud extrusion (Figure 1e), they were transferred to shaking culture to promote the growth of the tissue (Figure 1b and 1f). After 50 days of shaking culture in final differentiation media, the hMOs grew to approximately four millimeters (Figure 2a.b) and presented with several midbrain markers (Figure 2c). The presence of midbrain and dopaminergic markers were assessed by quantitative real-time qPCR. Compared to iPSC line sample, we observed an enrichment of several common midbrain markers (EN1, Nurr1, LMX1B, LMX1A, TH, MAOB, Calb1, DDC, COMT) in 50 days-old hMOs (Figure 2c). Conversely, we did not detect any enrichment for dopamine beta-hydroxylase as a marker for noradrenergic neurons (Figure 2c). Interestingly, by using SHH and FGF8 signals we also induce the formation of serotonergic neurons as detected by GBX2 marker (Figure 2c). This finding is consistent with recent reports with hMOs generated using other protocols 65 .
Immunostainings on cryosections of hMOs thirty-day old hMOs, showed a typical cytoarchitecture (Figure 3a) with multiple rosettes. The center of the rosettes was composed of neural progenitors cells, including Dopaminergic progenitors that were positive for FoxA2 (Figure 3b), negative for MAP2 (Figure 3b squares) while the outside layer is composed of more matured cells, including neurons that were MAP2 positive (Figure 3b arrows). As expected for hMos, we detected the presence of tyrosine hydroxylase (TH) cells (Figure 3c). This observation was confirmed by single cell RNA sequencing revealing dopaminergic lineage markers (Figure 3d-f). Unsupervised clustering yielded 8 clusters representing cell types that would be expected to be found in the human brain ( Figure 3d). The cell types mostly group together by unsupervised clustering. The cluster annotated as 'mixed' contains many cell types but few or no neurons. Radial glia are cells differentiated from stem cells into cells with the potential to become neurons or glia, Radial Glia-1 higher activation of ribosomal pathways than Radial Glia-2 which is further along the differentiation pathway. Neuronal cluster 1 contains interneurons, while cluster 2 contains high levels of dopaminergic markers (Figure 3e). The expression of the dopaminergic marker TH in Neurons -1, Neurons -2 and NPCs is 15%, 34% and 11.5% respectively. The average expression across the three clusters of neurons is 20% (Figure 3f).
Finally, we treated hMOs at day 35 with 100µM dopamine for 10 days to look at dopamine synthesis by-products with a focus on neuromelanin granules 66 . This experiment was not necessary for maturation of hMOs but allowed us to confirm the presence of dopaminergic neurons, as well as the ability of dopamine synthesis. We observed the appearance of brown/black areas, suspected to be neuromelanin granules accumulations, also known as by-products of dopamine synthesis (Figure 4a) 36 . Silver stains were shown to label neuromelanin granules in the substantia nigra 67 . Thus, we confirmed the presence of neuromelanin granules in hMOs by Fontana Masson staining (Figure 4b) and observed a significant increase of neuromelanin granules after dopamine treatment (Figure 4b-c). and its localisation in dopaminergic neurons (Figure 4d). Finally, we also generated hMOs from an iPSC line of patient with PD carrying triplication for synuclein (SNCA_Tri, Figure S1 b-c), and observed that they reached approximately 4 mm in size. Additionally, staining on 100 day-old SNCA_Tri revealed the presence of dopaminergic neurons too (TH) confirming the value of this protocol for future PD research studies. Raw images and data are available as underlying data.

Concluding remarks
In our group, we have successfully generated hMOs from patientderived iPSCs with similar dopaminergic neuron yield than previously published 36 . However, there are various challenges that are associated with the generation of organoids. (i) It is important to note that the quality of the iPSCs remains the most crucial step in the formation of organoid tissue. Differentiated iPSCs would either avoid proper formation of EBs or lead to the formation of non-homogenous EBs that would contribute to variable material for experimentations. iPSC lines are very sensitive and require delicate culture techniques to avoid differentiation. This can be achieved by choosing a range of iPSC passage number suitable for generating good EBs as well as spending extensive effort to remove any cell with differentiation sign. (ii) Batch-to-batch reproducibility is difficult to achieve. It can be controlled by optimizing chemical and physical parameters of media and incubation. (iii) Optimal concentrations of components in the media need to be carefully chosen. There are various chemical factors that contribute to the generation of organoids, and therefore require careful standardization.
(iv) Generation of uniform EBs, is the key factor in the generation of organoids. Uniform, smooth and continuous edges of EBs are essential to develop uniform organoids. The primary step for assessment is the neural induction that results in formation of embryoid bodies. The shape (spherical with smooth edges) of EBs at this stage is the defining factor of organoid formation. The EBs generated by our protocol have consistent shape. Although, to facilitate tissue induction and further develop a 3-dimensional structure, Matrigel® is used as a scaffold. Since this scaffold is present only in the early days of organoids, the shape until then is consistent due to this physical parameter in place whereas, once the organoids outgrow the scaffold and are capable of differentiating independently in the medium, the shape can vary slightly from one organoid to another (so far, we have observed slight variations in shape but not to a great extent). As the organoids are generated in a controlled environment, their shape and size is fairly consistent.
(v) The speed of the shaker is crucial in the final differentiation of organoids and maintaining 3D organization. (vi) The first protocols for cerebral organoids generation required used of paraffin, one-by-one Matrigel® embedding , and one-by-one transfer into final plates 34 . This procedure was time consuming and could led to tissue damaged or contamination by the multiple transfers steps involved. By using our midbrain generation protocol, we enabled a scaled-up production, without touching directly the tissue at any step. This allowed us to generate easily big batches of 500 hMOs derived from multiples iPSC lines for comparison studies. (vii) Cryosectioning organoids is a challenging analysis step. Since the organoids form a structure distinct from that of brain tissue, some protocol adaptations were necessary to consistently generate high quality sections. Furthermore, due to tissue organization and small sizes, sections have to be optimized for each stage of organoid maturation. Other methods such as clearing techniques can be useful to overcome this challenge. So far, we have overcome the challenges and generated numerous high-quality midbrain organoids. This method manuscript is aiming to help the community to generate hMOs for Parkinson's disease studies. Moreover, it provides interesting information on the cell composition of the organoids, highlighting the presence of glial clusters. A few additional minor improvements will make this review a valuable contribution helping people in the field to generate human midbrain organoids in a straightforward manner.

Data availability
Minor points: The photographs in Figure 3b-c are small and it remains difficult to appreciate the full morphology of the neurons (cell bodies and processes). It would be nice if the authors could provide higher magnifications for the images in panels b and c. 1.
In Figure 4d it is hardly possible to discriminate between the dark-green neuromelanin and the supposedly dark-brown TH staining.

2.
In Figure 2c, expression levels of the various markers in hMO are indicated comparatively to iPSC. Please indicate the age of the iPSC culture in the corresponding figure legend (days in vitro).

3.
The sentence "we detected serotonergic neurons marker (GBX2)…" should be modified. GBX2 expression indicates that markers of the serotonigergic lineage are expressed, but you would still need tryptophan hydroxylase 2 (TPH2) to get serotonin synthesis.

4.
"This procedure was time consuming and could led (lead) to tissue damaged (damage) or contamination by the multiple transfers (transfer) steps involved." Please correct.

5.
Competing Interests: No competing interests were disclosed.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. It is important to advance protocols for robust differentiation into 3D cultures. Overall, it is a comprehensive and detailed protocol. The manuscript is lacking is a section on troubleshooting.
Since the protocol is geared towards disease research, it would be critical to also include disease cell lines in the protocol (e.g. genetic forms of Parkinson's disease) and describe to what extent differences in the differentiation potential are evident and how to overcome them. No data is shown for cytoarchitecture for 50 days organoids. Is the center becoming necrotic in these larger organoids? Please describe and include.
Page 5, second paragraph: replace "vitamin B27 without vitamin A" with "B27 supplement without vitamin A".
Page 7, second paragraph: iPSC lines, especially from a repository can vary widely in passage number between early 10's to 50's and 60's, e.g. after editing. It seems arbitrary to write "10 passages". Please clarify.
Page 7, last paragraph: change "anti-anti" to "antibiotic-antimycotic", also correct spelling in Table  1 and footnote of Table 3 accordingly.
Page 8, timeline b.: panel below days: first two boxes should be deleted, also please what is the difference between "tissue growth" and "organoid growth". The timeline ends with several months, but organoids are only described macroscopically until day 50 and microscopically until day 35.
Page 8, legend: It is noted that PBMCs were reprogrammed, however, table 7 describes the commercial source of the iPSCs. Please clarify.
Page 13, second paragraph: Re-write sentence starting with "Silver stains…" Sentence structure and context is not clear.

Is the description of the method technically sound? Yes
Are sufficient details provided to allow replication of the method development and its use by others? Yes

If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly Competing Interests: No competing interests were disclosed.
Reviewer Expertise: stem cell modeling, Parkinson's research I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 06 Jan 2021

nguyen-vi mohamed, Montreal Neurological Institute and Hospital, Montreal, Canada
It is important to advance protocols for robust differentiation into 3D cultures. Overall, it is a comprehensive and detailed protocol. The manuscript is lacking is a section on troubleshooting. Since the protocol is geared towards disease research, it would be critical to also include disease cell lines in the protocol (e.g. genetic forms of Parkinson's disease) and describe to what extent differences in the differentiation potential are evident and how to overcome them.No data is shown for cytoarchitecture for 50 days organoids. Is the center becoming necrotic in these larger organoids? Please describe and include.

Answer:
We thank the reviewer for these comments. We agreed that the ultimate purpose of generating midbrain organoids is to conduct studies in Parkinson's disease (PD) hMOs.
Nonetheless, the precise analysis of differences between healthy hMOs or isogenic corrected hMOs compared to PD hMOs is beyond the scope of this manuscript which aims to generate hMOs from iPSC lines. Since we agreed that it's interesting to show that such comparison would be possible in future disease research manuscript, we included new cytoarchitecture data on PD hMOs derived from an iPSC line from a patient carrying a triplication for synuclein (SNCA_Tri) (  Figure 3c and Figure S1b-c). In Figure S1b, SNCA_Tri hMOs reached approximatively 4mm in diameter. In Figure 3c you can observe cryosections of 30 day-old SNCA_Tri hMOs, while in Figure S1c, you can observe a 100 day-old section from SNCA_Tri hMOs, both stained for TH (red) and MAP2 (green). We complemented the results section accordingly. We also would like to point out that troubleshooting sections are the "NOTE" points. Since another reviewer made a complementary comment for deeper troubleshooting details, we either added a "NOTE" or added more details to the pre-existing one.
Minor points: Page 3, last paragraph: define "generation rate of near 100%". Mohamed et. al. describes an optimized protocol to generate human midbrain organoids derived from human iPSCs. In this article, the authors utilize multiple hiPSC lines and optimize chemical and physical parameters of media formulations and incubation times to achieve a standardized protocol. This manuscript is very well written and contains many critical details necessary for successful organoid generation and culture. There are a number of minor details that I highlight below that when incorporated would increase the quality and rigor of the current manuscript.
In the background section, paragraph 2: "Since their discovery, this technology has opened up many new research avenues, including for PD." Please restructure this sentence to the following: "Since their discovery, this technology has opened up many new avenues of investigation, including research for PD." 1.
In the materials section, first paragraph, first sentence: "The materials, reagents and equipment listed in this document can be substituted" should be restructured and written: "The materials, reagents and equipment listed in this document can be substituted for comparable items."

3.
Mention of lot to lot variability is important and those items should designated with an asterisk in Table 1 and defined in the text.

4.
In the materials section describing NIM, "the help of" can be deleted. 5.
On page 9, after describing the orbital shaking speed of 70rpm, there should be another sentence following stating the rpm is dependent upon the type of shaker utilized that is based on the throw (shaking diameter) of the shaker.

6.
Since two hiPSC lines were tested using this protocol, some details should be given describing whether both lines achieved similar efficiencies or not. In addition, details on how many independent experiments were performed to achieve the optimized parameters should be mentioned.

Is the description of the method technically sound? Yes
Are sufficient details provided to allow replication of the method development and its use by others? Partly Mohamed et. al. describes an optimized protocol to generate human midbrain organoids derived from human iPSCs. In this article, the authors utilize multiple hiPSC lines and optimize chemical and physical parameters of media formulations and incubation times to achieve a standardized protocol. This manuscript is very well written and contains many critical details necessary for successful organoid generation and culture. There are a number of minor details that I highlight below that when incorporated would increase the quality and rigor of the current manuscript.
We thank Dr Hester for his approval and insightful comments. Please find attached a revised version of the manuscript and below our replies to the points raised.
In the background section, paragraph 2: "Since their discovery, this technology has opened up many new research avenues, including for PD." Please restructure this sentence to the following: "Since their discovery, this technology has opened up many new avenues of investigation, including research for PD." ○ Answer: Thanks to the reviewer's comments, we have now adjusted the sentence as recommended.
In the background section, column 2, paragraph 4: "for a robust derivation of iPSCs ○ lines into 3D midbrain…" "iPSCs" should be singular and read "iPSC lines into 3D…" Answer: Thanks to the reviewer's comments, we have now adjusted the sentence as recommended. We also corrected the typo thought the manuscript "The observations provided in this method were generated with at least 6 independent batches derived from two iPSC lines from healthy individuals (NCRM-1 and XCl-1) or an iPSC line from a patient with PD (EDI001A named as SNCA_Tri in Figures) (Table 7)." / "Human iPSC lines are to be handled within a Class II biosafety laminar flow hood to protect the worker from possible biohazardous agents." In the materials section, first paragraph, first sentence: "The materials, reagents and equipment listed in this document can be substituted" should be restructured and written: "The materials, reagents and equipment listed in this document can be substituted for comparable items." ○ Answer: Thanks to the reviewer's comments, we have now adjusted the sentence as recommended.
Mention of lot-to-lot variability is important and those items should designated with an asterisk in Table 1 (Table 6)." Since two hiPSC lines were tested using this protocol, some details should be given describing whether both lines achieved similar efficiencies or not. In addition, details on how many independent experiments were performed to achieve the optimized parameters should be mentioned.  (Table 7)". Additionally, we added a representative picture of XCl-1 derived hMOs, 50-day aged respectively, to show similar efficiency achievement (Figure 2b). We updated the legends accordingly. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Author Response 06 Jan 2021 nguyen-vi mohamed, Montreal Neurological Institute and Hospital, Montreal, Canada The manuscript by Nguyen-Vi Mohamed and colleagues describes a standardized procedure for the generation and characterization of midbrain organoids from human iPSC lines, developed by adapting and integrating key reference protocols in the field. The production of such organoids is of great importance for studying human brain development and modeling neurodevelopmental and neurodegenerative diseases, in particular Parkinson's disease. The manuscript is well written and the experimental procedures are described in great detail, nicely pinpointing key steps that require special attention and experimental tricks to help perform the experiments under ideal and controlled conditions. Following the provided procedure, any personnel skilled in stem cell culture should be able to easily generate human midbrain organoids in an appropriate environment. The protocol may be improved to ensure a more complete characterization of the quality of the midbrain organoids, so as to fully support the conclusions drawn and increase the impact of this paper.
Major points: The authors built on previous work and the full characterization of the dopaminergic neurons present in the organoids is probably beyond the scope of this manuscript. Nevertheless, it would be useful to provide staining for other basic markers to confirm the efficacy and specificity of the differentiation procedure and exclude the presence of other neuronal types that also express the TH marker. The authors may consider checking for: (i) other enzymes of the dopamine biosynthesis pathway, such as GTP cyclohydrolase 1 or DOPA decarboxylase, and the dopamine transporter; (ii) as well as dopamine beta-hydroxylase, as a marker for noradrenergic/adrenergic neurons.

1.
Testing for a few midbrain markers would be useful to fully support the generation of midbrain organoids. The standard protocol used to pattern ventral midbrain structures is based on the combination of SHH and Fgf8 signals and it is important to keep in mind that this combination patterns both ventral midbrain and ventral hindbrain in the embryo. For example, both midbrain dopaminergic and hindbrain serotonergic neurons are dependent on these induction signals. It would therefore be of interest to evaluate whether other, more rostral or more caudal brain areas, are also represented in these organoids. A broader characterization of the generated cell types could be achieved by QPCR marker expression analysis and a few additional immunostainings.

2.
Answer: We thank the reviewer for raising these two points. We added a complementary qRT-PCR experiment in Figure 2cshowing (Figure 3d-f) and revealed the presence of others cell types that includes astrocytes, oligodendrocyte precursors, radial glial, neuronal progenitors as reported by other groups (35,65). A discussion of the significance of the achievements with respect to previous publications would be useful. In how far do the adaptations to previous protocols proposed here improve the reproducibility or robustness of the approach? The authors mention that the quality of the iPSCs is critical and that"variability in the iPSCs maintenance will negatively impact midbrain organoid generation". What do they mean by quality and variability, and what kind of negative impact is expected?  Figure 4d) (64). The neuromelanin changes under dopamine treatment were analyzed using GIMP software to specifically extract the pixels associated with neuromelanin staining from of Fontana-Masson-stained sections and quantitating the number of extracted pixels using Image J (63). A histogram of the image was created, which separates the total number of pixels in the image into 255 color categories spanning the visible spectrum. The peak corresponding to the brown-black color (neuromelanin) was determined by cutting and summing the appropriate counts from each channel of the melanin peak. In order to clarify the graph, we provided more quantification details in methods as follow: "Black dots from Fontana Masson stainings pictures were extracted with colorimetric selection from GIMP software (version 2.8.22) and quantified by ImageJ (version 2.0.0-rc-69/1.52i) following the method described in (63). Briefly, using GIMP software the pixels associated with neuromelanin staining were colored extracted, and quantitating the number of extracted pixels using Image J. A histogram of the image was created, which separates the total number of pixels in the image into 255 color categories spanning the visible spectrum. The peak corresponding to the brown-black color i.e. neuromelanin was determined by cutting and summing the appropriate counts from each channel of the melanin peak." Additionally, since we agreed that the neuromelanin numbers were not a direct quantification of the neuromelanin but rather a black pixels quantification, we modified the title axis for "Relative number of neuromelanin granules" in Figure 4c.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
this step necessary for maturation (i.e. neuromelanin production)? Do the organoids release dopamine exogenously or are the only capable or metabolizing exogenous dopamine. Does this step introduce any challenges with modeling aspects of PD where DA-quinone production is considered a toxic insult?
Answer: In Figure 4, we treat the young organoids with dopamine to trigger dopaminergic metabolism and observe the formation of neuromelanin granules, as a confirmation for the presence of dopaminergic neurons and their functionality. This step is not necessary for maturation of hMOs. We routinely perform this assessment, on several hMOs from every batch produced, as a tool to quickly confirm the midbrain identity of organoids produced. When we age the hMOs to within 5-6 months, we observe in the absence of any external dopamine treatment, a spontaneous formation of neuromelanin granules meaning the hMOs release dopamine exogenously. We thank the reviewer for his comment, and we clarified this point in the result section to avoid future confusion: "This experiment was not necessary for maturation of hMOs but allowed to confirm the presence of dopaminergic neurons, as well as the ability of dopamine synthesis." 7-Can the authors comment on the consistency in the shape of their organoids as opposed to size alone. Does the shape correlate with the quality of the differentiation or variability in differentiation?
Answer: The primary step for assessment is the neural induction that results in formation of embryoid bodies. The shape (spherical with smooth edges) of EBs at this stage is the defining factor of organoid formation. The EBs generated by our protocol have consistent shape. Although, to facilitate tissue induction and further develop a 3-dimensional structure, Matrigel® is used as a scaffold. Since this scaffold is present only in the early days of organoids, the shape until then is consistent due to this physical parameter in place whereas, once the organoids outgrow the scaffold and are capable of differentiating independently in the medium, the shape can vary slightly from one organoid to another (so far, we have observed slight variations in shape but not to a great extent). As the organoids are generated in a controlled environment, their shape and size are fairly consistent. We thank the reviewer for his comment, and we added this point in the conclusion remarks.
Competing Interests: No competing interests were disclosed.