This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.
The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.
The emergence of human pluripotent stem cells (PSCs) represents a excellent tool for regenerative medicine and disease modeling, because these cells can be differentiated into most cell lineages of the human body1,2. Since their discovery, PSC differentiation using diverse approaches has been reported to model different diseases, including neurodegenerative disorders3,4,5,6.
Recently, there have been reports of 3D cultures derived from PSCs resembling human cerebral structures; these are called brain organoids3,7,8. The generation of these structures from both healthy and patient-specific PSCs provides a valuable opportunity to model human development and neurodevelopmental disorders. However, the methods used to generate these well-organized cerebral structures are difficult to apply for their large-scale production. To produce structures that are large enough to recapitulate tissue morphogenesis without necrosis inside the organoids, protocols rely on the initial neural commitment in static conditions, followed by encapsulation in hydrogels and subsequent culture in dynamic systems3. However, such approaches may limit the potential scale-up of organoid production. Even though efforts have been made to direct PSC differentiation to specific regions of the central nervous system, including cortical, striatal, midbrain, and spinal cord neurons9,10,11,12, the generation of specific brain regions in dynamic conditions is still a challenge. In particular, the generation of mature cerebellar neurons in 3D structures has yet to be described. Muguruma et al. pioneered the generation of culture conditions that recapitulate early cerebellar development13 and recently reported a protocol that allows for human embryonic stem cells to generate a polarized structure reminiscent of the first trimester cerebellum7. However, the maturation of cerebellar neurons in the reported studies requires the dissociation of the organoids, sorting of cerebellar progenitors, and coculture with feeder cells in a monolayer culture system7,14,15,16. Therefore, the reproducible generation of the desired cerebellar organoids for disease modeling under defined conditions is still a challenge associated with culture and feeder source variability.
This protocol presents optimal culture conditions for 3D expansion and efficient differentiation of human PSCs into cerebellar neurons using single-use vertical wheel bioreactors (see Table of Materials for specifications), hereafter called bioreactors. Bioreactors are equipped with a large vertical impeller, which in combination with a U-shaped bottom, provide a more homogeneous shear distribution inside the vessel, allowing gentle, uniform mixing and particle suspension with reduced agitation speeds17. With this system, shape and size-controlled cell aggregates can be obtained, which is important for a more homogeneous and efficient differentiation. Moreover, a larger number of iPSC-derived organoids can be generated in a less laborious manner.
The main feature of the organoids, which are 3D multicellular structures usually formed from stem cells, is the self-organization of different cell types that forms specific shapes like those seen in human morphogenesis18,19,20. Therefore, organoid morphology is an important criterion to be evaluated during the differentiation process. Cryosectioning of organoids and further immunostaining of organoid slices with a specific set of antibodies allow for the spatial visualization of molecular markers to analyze cell proliferation, differentiation, cell population identity, and apoptosis. With this protocol, by immunostaining organoid cryosections, an initial efficient neural commitment is observed by the 7th day of differentiation. During differentiation, several cell populations with cerebellar identity are observed. After 35 days in this dynamic system, the cerebellar neuroepithelium organizes along an apicobasal axis, with an apical layer of proliferating progenitors and basally located postmitotic neurons. During the maturation process, from days 35–90 of differentiation, distinct types of cerebellar neurons can be seen, including Purkinje cells (Calbindin+), granule cells (PAX6+/MAP2+), Golgi cells (Neurogranin+), unipolar brush cells (TBR2+), and deep cerebellar nuclei projection neurons (TBR1+). Also, a nonsignificant amount of cell death is observed in the generated cerebellar organoids after 90 days in culture.
In this system, human iPSC-derived organoids mature into different cerebellar neurons and survive for up to 3 months without the need for dissociation and feeder coculture, providing a source of human cerebellar neurons for disease modeling.
1. Passaging and maintenance of human iPSCs in monolayer culture
2. Seeding of human iPSCs in the bioreactor
3. Differentiation and maturation of human iPSC-derived aggregates in cerebellar organoids
4. Preparation of organoids for cryosectioning and immunohistochemistry
The protocol was initiated by promoting cell aggregation using the 0.1 L bioreactors (Figure 1A). Single cell inoculation of the iPSCs was performed, with 250,000 cells/mL seeded in 60 mL of medium with an agitation speed of 27 rpm. This was defined as day 0. After 24 h, the cells efficiently formed spheroid-shaped aggregates (day 1, Figure 1B), and the morphology was well-maintained until day 5, with a gradual increase in size, demonstrating a high degree of homogeneity in aggregate morphology and size over time. (Figure 1B). A quantitative analysis by microscopy also revealed normal distribution of aggregate sizes by day 1 (Figure 1C). The aggregate size is an important physical parameter capable of prompting the cells to differentiate toward different lineages29,30. For this reason, based on the aggregate size reported in previous studies to induce an efficient neural31,32 and cerebellar commitment21, the generated aggregates were maintained in mTeSR1 medium at 25 rpm until they reached the desired diameter before starting differentiation (~200 µm). At day 2, the average diameter was 221.0 ± 54.4 µm (mean ± SD) for the F002.1A.13 cell line and 212.1 ± 42.1 µm for the iPSC6.2 cell line. As such, both cell lines attained the optimal aggregate size at this timepoint (Figure 1C).
Defining the day on which the seeding of iPSCs was performed as day 0, at day 2, after achieving the desired aggregate diameter, neural commitment was induced by simultaneously using SB431542, FGF2, and insulin, promoting neuroectodermal differentiation, as well as a moderate caudalization necessary for mid-hindbrain patterning. Afterwards, FGF19 and SDF1 were added to the culture at days 14 and 28, respectively, to promote the generation of different cerebellar progenitors. For the first days of neural induction, a rotation speed of 25 rpm was used, which was increased to 30 rpm after 7 days to avoid the accumulation and clumping of bigger aggregates (Figure 2A). During differentiation, organoids showed a more pronounced epithelization similar to neural tube-like structures with luminal space (Figure 2B). Additionally, the evaluation of organoid diameter distribution demonstrated a homogeneous size distribution during the initial cerebellar commitment until day 14 (Figure 2B).
Immunofluorescence analysis supports that an efficient neural commitment of the iPSC-derived organoids is already achieved by day 7 of differentiation after adding FGF2 and SB431542. The cryosections of organoids revealed many structures reminiscent of the neural tube staining for PAX6 and NESTIN, with most cells within the organoids expressing progenitor marker NESTIN at days 7 and 14 of differentiation (Figure 2C). Afterwards, FGF19 and SDF1 promoted the generation of continuously proliferating progenitor layers (PAX6+) and an efficient neuronal differentiation was achieved, as demonstrated by the expression of TUJ1, neuron-specific class III beta-tubulin, by days 21 and 35 (Figure 2C). In addition, an efficient cerebellar differentiation was also observed after 21 days in the 0.1 L VW bioreactors, demonstrated by the presence of two different cell populations: granule cell progenitors (BARLH1+ cells, Figure 3A), and Purkinje cell progenitors (OLIG2+ cells, Figure 3B). After 35 days in culture, different cell populations within the organoids appeared to be organized into distinct layers. Various flat-oval structures within the organoids were observed with BARHL1+ dorsal cerebellar progenitors as a continuous layer on the superficial side of the organoid (Figure 3C,D) and SOX2+ in the luminal region of these oval structures (Figure 3D). In addition, TUJ1+ newborn neurons appeared to migrate towards the surface, reestablishing the radial alignment on the outer surface of the organoid (Figure 3E).
After the generation of cerebellar progenitors, further maturation was promoted using BrainPhys medium28 supplemented with neurotrophic factors BDNF and GDNF. Immunofluorescence staining of organoid cryosections was used to detect distinct subtypes of cerebellar neurons. Purkinje cells, GABAergic neurons expressing the calcium-binding protein calbindin (CALB, Figure 3F), were detected in the cerebellar organoids after the maturation protocol. In addition, another major cerebellar neuronal type, granule cells, was identified as a subset of cells coexpressing PAX6 and MAP2 (Figure 3G). Interestingly, a pool of PAX6+ progenitors not expressing MAP2 was maintained until 80 days of differentiation. Other types of cerebellar neurons were also detected, including unipolar brush cells expressing TBR2 (Figure 3H), and deep cerebellar nuclei projection neurons expressing TBR1 (Figure 3I). In addition to efficient cerebellar differentiation and maturation, this 3D dynamic culture system using the PBS 0.1 L VW bioreactors allowed organoids to remain viable for up to 90 days, without significant cell death and necrosis (Figure 3J).
Figure 1: Generation of size-controlled aggregates using scalable bioreactors. (A) Design features of the bioreactor. (B) Brightfield photomicrograph showing aggregates from two different iPSC lines on days 1, 2, and 5. Scale bar = 100 μm. (C) The size distribution of floating aggregates from different iPSC lines in the bioreactors. Please click here to view a larger version of this figure.
Figure 2: Generation of human iPSC-derived organoids using 0.1 L bioreactors. (A) Schematic representation of the culture procedure to induce differentiation of iPSCs to cerebellar organoids. Cells were seeded at a density of 250,000 cells/mL and an agitation speed of 27 rpm was used to promote cell aggregation. During the first days of differentiation, aggregates were maintained at an agitation speed of 25 rpm. Afterwards, to avoid the accumulation of bigger aggregates, the agitation speed was increased to 30 rpm. (B) Characterization of organoid shape and size. Brightfield photomicrographs showing iPSC-derived organoids during cerebellar differentiation in the 0.1 L VW bioreactors. Scale bar = 100 μm. The distribution of organoid diameters demonstrates that the culture maintained homogeneous organoids sizes along the differentiation protocol. (C) Efficient neural induction in iPSC-derived organoids. Immunofluorescence for NESTIN, PAX6, and TUJ1 during cerebellar differentiation. Scale bar = 50 μm. Please click here to view a larger version of this figure.
Figure 3: Efficient cerebellar differentiation and maturation in human iPSC-derived organoids. (A–E) Efficient cerebellar commitment. Immunostaining analysis for BARHL1, SOX2, OLIG2, NCAD, and TUJ1 markers at indicated timepoints of the cerebellar differentiation protocol. (F–I) Efficient maturation of human iPSC-derived cerebellar organoids. Immunofluorescence showing different types of cerebellar neurons, including Purkinje cells (CALB, F), granule cells (PAX6 and MAP2, G), unipolar brush cells (TBR2), and deep cerebellar nuclei projections neurons (TBR1). (J) High cell viability after cerebellar maturation. Live/dead (calcein-AM, green and propidium iodide, red) staining of organoids showed high cell viability and no evidence of necrotic areas after 80 days in the bioreactors. Scale bar = 50 μm. Please click here to view a larger version of this figure.
Media preparation | mTeSR1 Final volume: 500 mL |
1. Thaw mTeSR1 5× supplement at room temperature (RT) or at 4 °C overnight and mix with basal medium 2. Store complete mTeSR1 medium at 4 °C for up to 2 weeks or prepare 40 mL aliquots and store at -20 °C 3. Pre-warm complete mTeSR1 at RT before use |
gfCDM (growth factor-free chemically defined medium) Final volume: 60 mL |
30 mL Ham’s F12 30 mL IMDM 600 µL chemically defined lipid concentrate (1 % v/v) 2.4 µL monothioglycerol (450 μM) 30 µL apo-transferrin (stock solution at 30 mg/mL in water, final concentration: 15 μg/mL) 300 mg crystallization-purified BSA (5 mg/mL) 42 µL insulin (stock concentration at 10 mg/mL, final concentration: 7 µg/mL) 300 µL P/S (0.5% v/v, 50 U/ml penicillin/50 μg/ml streptomycin) |
|
Neurobasal Final volume: 60 mL |
60 mL of Neurobasal medium 600 µL N2 supplement 600 µL Glutamax I 300 µL P/S (0.5 % v/v). |
|
Complete BrainPhys Final volume: 60 mL |
60mL of BrainPhys 1.2 mL NeuroCult SM1 Neuronal Supplement 600 µL N2 Supplement 12 µL BDNF (final concentration: 20 ng/mL) 12 µL GDNF (final concentration: 20 ng/mL) 300 µL Dibutyryl-cAMP (stock concentration: 100 mg/mL in water, final concentration: 1 mM) 42 µL ascorbic acid (stock concentration: 50 µg/mL in water, final concentration: 200 nM) |
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Stock solutions of growth factors and small molecules | Basic fibroblast growth factor (bFGF/FGF2) Stock concentration: 100 µg/mL |
1. Reconstitute in 5 mM Tris, pH 7.6, at a concentration of 10 mg/mL 2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 µg/mL |
Stromal cell-derived factor 1 (SDF1) Stock concentration: 100 µg/mL |
1. Reconstitute in water at a concentration of 10 mg/mL 2. Dilute with 0.1 % BSA (v/v) in PBS to a final stock concentration of 100 µg/mL. |
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Brain-derived neurotrophic factor (BDNF) Stock concentration: 100 µg/mL |
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Glial cell-derived neurotrophic factor (GDNF) Stock concentration: 100 µg/mL |
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Fibroblast growth factor 19 (FGF19) Stock concentration: 100 µg/mL |
1. Reconstitute in 5 mM sodium phosphate, pH 7.4, at a concentration of 10 mg/mL 2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 µg/mL |
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ROCK inhibitor Y-27632 Stock concentration: 10mM |
Reconstitute in DMSO at a concentration of 10 mM. | |
SB431542 Stock concentration: 10mM |
||
Insulin Stock concentration: 10 mg/mL |
1. Reconstitute 10 mg of insulin in 300 µL of 10 mM NaOH 2. Carefully add 1 M NaOH until the solution becomes clear-transparent 3. Fill to 1 mL with sterile water. |
Table 1: Stock solutions and media preparation. Listed are all the components and volumes used to prepare media for the iPSCs maintenance and differentiation protocol, as well as stock solutions of growth factors and small molecules. For stock solutions, all stock concentration and protocols for reconstitution are listed.
Gelatin/Sucrose Final concentration: 7.5%/15% w/w |
1. Weigh 15 g of sucrose and 7.5 g of gelatin in a sterile Schott Glass Bottle and mix well 2. Pre-warm the PBS 1× at 65 °C 3. Add pre-warmed PBS 1× to a final weight of 100 g and mix well 4. Place the Schott Glass Bottle in a heating plate at 65 °C and shake until the gelatin melts 5. Incubate at 37 °C until the solution stabilizes |
Glycine Final concentration: 0.1 M |
Add 0.37 g glycine to 50 mL of freshly-prepared PBS 1×. |
Triton solution Final concentration: 0.1 % w/v |
1. Prepare a 10 % Triton X-100 stock: 5 g of Triton X-100 in 50 mL of PBS 1× 2. Add 0.5 mL of Triton X-100 stock to 50 mL of PBS 1×. |
TBST 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05 % w/v Tween-20 |
20 mL Tris 1 M 30 mL NaCl 5 M 5 mL Tween-20 (10 % stock: 5 g of Tween-20 in 50 mL water) Fill to 1 L with water. |
Blocking Solution | Add 5 mL of fetal bovine serum (FBS, final concentration: 10 % v/v) to 50 mL of TBST. |
DAPI solution | Add 15 µL of DAPI stock solution (1 mg/mL) to 10 mL of destilated water |
Mowiol | 1. Add 2.4 g of Mowiol to 6 g of glycerol and shake for 1 h in a pre-warmed plate at 50 °C 2. Add 6 mL of distilled water and shake for 2 h 3. Add 12 mL of Tris 200 mM (pH 8.5) and shake for 10 min 4. Centrifuge at 5,000 × g for 15 min 5. Aliquot and store at -20 °C. |
Table 2: Solutions for preparation of organoids for cryosectioning and immunostaining. Listed are all the components and volumes used to prepare the solutions used in the preparation of organoids for cryosectioning and immunostaining.
Antibody | Host species | Dilution |
BARHL1 | rabbit | 1/500 |
CALBINDIN | rabbit | 1/500 |
MAP2 | mouse | 1/1000 |
N-CADHERIN | mouse | 1/1000 |
NESTIN | mouse | 1/400 |
OLIG2 | rabbit | 1/500 |
PAX6 | rabbit | 1/400 |
SOX2 | mouse | 1/200 |
TBR1 | rabbit | 1/200 |
TBR2 | rabbit | 1/200 |
TUJ1 | mouse | 1/1000 |
Table 3: Primary antibodies. The primary antibodies, clone, and optimized dilutions used for immunostaining are listed.
The need for large cell numbers as well as defined culture conditions to generate specific cell types for drug screening and regenerative medicine applications has been driving the development of scalable culture systems. In recent years, several groups have reported the scalable generation of neural progenitors and functional neurons32,33,34, providing significant advances in the development of new models for neurodegenerative disorders. Nonetheless, the recapitulation of some critical events of embryonic development is still lacking, and the maintenance of the generated functional neurons in suspension for long periods of time has not yet been achieved34. Presented here is a dynamic 3D culture system able to generate iPSC-derived neural organoids with cerebellar identity, and to further promote maturation into functional cerebellar neurons under chemically-defined and feeder-free conditions in dynamic culture.
Before starting cerebellar differentiation, it is critical to maintain the quality of the human iPSCs. Thus, in order not to compromise the differentiation, no more than three passages of iPSCs should be performed from thawing to bioreactor inoculation. An important step in the differentiation protocol is to evaluate the aggregate size. The aggregate size has a critical role in inducing differentiation towards a specific cell lineage29. Besides that, there is a minimum size threshold that appears to favor differentiation35. As already reported, the optimal iPSC-derived aggregate diameter to promote an efficient neural commitment31,32 and cerebellar differentiation21 is a ~200 µm diameter.
Additionally, in this dynamic protocol, the agitation speed used in the first days of culture is crucial to control the aggregate diameter and neural induction. The culture started at 27 rpm, which is sufficient to promote iPSCs aggregation and to avoid the formation of larger aggregates (diameters above 350 µm should be avoided). The agitation used to promote cell aggregation after single cell seeding could be increased to 30 rpm without affecting cell viability; however, higher agitation speeds are expected to produce smaller aggregates. Depending on the iPSC line, 24 h after cell seeding using 27 rpm, two different scenarios are expected: the aggregates formed present smaller diameters (<200 µm) or have reached a range of sizes between 200–300 µm. If aggregates are larger than 350 µm at 24 h after cell seeding, differentiation should not be performed, and the cell seeding should be repeated, because the efficiency of the differentiation will be very low. If aggregates are smaller than 200 µm, the spent medium should be replaced with iPSC maintenance medium, and the agitation speed reduced to 25 rpm. With this adjustment, aggregate diameter is expected to increase from day 1 to day 2, probably due to the merging of individual aggregates promoted by the decrease in the agitation speed. In case of aggregates with sizes between 200–300 µm, the spent medium should be replaced with differentiation medium, and neural induction with FGF2 should be started after 2 days in culture. At this point, the agitation speed should also be slightly reduced to prevent excessive cell death, because cells are more sensitive to shear stress in the presence of differentiation medium. Additionally, the population homogeneity could be analyzed using the coefficient of variation (CV), which measures the variability by correlating standard deviation with the mean of aggregate diameters, according to the equation
in which δ represents the standard deviation of the aggregate diameter and μ is the average diameter. In this dynamic system, the observed average CV was 12.5 ± 3.3% (mean ± SD) for the F002.1A.13 cell line and 19.0 ± 0.37% for the iPSC6.2 cell line at day 2. Thus, in this system, a homogeneous size population with a CV below 0.2 (< 20% of variation) should be expected. After 7 days of differentiation, the average aggregate diameter ranged from 300–360 µm, and the agitation speed was increased to 30 rpm to prevent aggregates to settle at the bottom of the 0.1 L VW bioreactor.
The differentiation of cerebellar organoids until day 35 and the analysis of aggregate size in static conditions were recently reported21. The authors showed that 3D aggregates formed and maintained in plates (e.g., Aggrewell) until day 7 of differentiation were homogeneous in size and shape21. However, after transferring the aggregates to ultra-low attachment 6 well culture plates, the aggregates started to vary in size and morphology21. On day 35 in static conditions, some of the 3D aggregates reached 1,000 μm for different cell lines, which limited the diffusion of nutrients and oxygen. In contrast, using our dynamic conditions, aggregates did not reach more than 800 μm in diameter by day 35, with improved mass transfer due to the constant agitation of the medium promoted by the vertical wheel. Furthermore, the aggregate sizes were maintained until the end of the maturation process, showing an aggregate diameter of 646.6 ± 104.2 μm by day 90, the longest culture performed in 0.1 L VW bioreactors.
Efficient cerebellar induction was induced by sequential addition of SB431542, FGF2, FGF19, and SDF1 in this 3D dynamic system. The protocol starts with the combination of SB431542, which is a transforming growth factor beta (TGF-ß)-receptor blocker that inhibits mesendodermal differentiation, and FGF2, which has a major effect in the caudalization of neuroepithelial tissue25. Therefore, the addition of these two molecules during the first days of culture is essential to promote the cell differentiation to the mid-hindbrain, the territory that gives rise to the cerebellar tissue. After initial induction to mid-hindbrain tissue, it is necessary to add FGF19 for promoting the spontaneous generation of mid-hindbrain structures with dorsal-ventral polarity, as well as the generation of different cerebellar progenitors36,26. SDF1 facilitates the organization of distinct layers of cerebellar progenitors, as seen at the developmental stage in which cerebellar neurogenesis occurs27. Until day 35, these molecules can promote the organization of cerebellar organoids that can recapitulate human cerebellar development, which corresponds to the first trimester cerebellum. After the organization of cerebellar progenitors into different layers, a defined neuronal medium was used to promote their maturation28. Other media used to maintain neuronal cells could also be tested, but lower efficiencies are anticipated. Thus, in this protocol, BrainPhys was used to promote the differentiation of cerebellar-committed cells into cerebellar neurons, because it has been reported to better mimic the healthy neuronal environment and to support neurophysiological activity of the generated neurons28.
Using these dynamic conditions, a more efficient diffusion of nutrients, oxygen, and growth factors can be achieved. However, some limitations are associated with the agitation used in the differentiation protocol. Some shear stress can be introduced by the agitation process, which can affect the survival, proliferation, and differentiation of cells. Therefore, during the maturation step, in which the cells are more sensitive, the culture must be carefully monitored.
The differentiation of cerebellar organoids reminiscent of human embryonic cerebellar development has already been reported7. However, further maturation of these embryonic cerebellar organoids into cerebellar neurons using 3D cultures remains a challenge. The generation of functional cerebellar neurons was only achieved by coculturing with granule cells from various sources4,7,15. This protocol successfully upscaled cerebellar commitment of human iPSCs; in addition, this is the first protocol for the differentiation of different cerebellar neurons in a 3D culture system without coculturing with feeder cells. Specifically, the following cell types can be produced in our dynamic culture system: Purkinje cells (Calbindin+), granule cells (PAX6+/MAP2+), unipolar brush cells (TBR2+), and deep cerebellar nuclei projection neurons (TBR1+), which were maintained in suspension for as long as 3 months.
The scalable generation of cerebellar organoids represents a valuable tool for studying the embryonic development of the cerebellum and the pathological pathways involved in the degeneration of this organ. Furthermore, high-throughput screening for molecules that restore cerebellar function may be performed using organoids obtained with this scalable system. Overall, this method satisfies an unmet need for a scalable protocol for the generation of high-quality cerebellar organoids that may be important for a variety of biomedical applications.
The authors have nothing to disclose.
This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project N. 007317, PD/BD/105773/2014 to T.P.S and PD/BD/128376/2017 to D.E.S.N.), projects co-funded by FEDER (POR Lisboa 2020—Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research grant LISBOA-01-0145-FEDER-029298. Funding was also received from the European Union's Horizon 2020 Research and Innovation Programme, under the Grant Agreement number 739572—The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017.
3MM paper | WHA3030861 | Merck | |
Accutase | A6964 – 500mL | Sigma | cell detachment medium |
Anti-BARHL1 Antibody | HPA004809 | Atlas Antibodies | |
Anti-Calbindin D-28k Antibody | CB28 | Millipore | |
Anti-MAP2 Antibody | M4403 | Sigma | |
Anti-N-Cadherin Antibody | 610921 | BD Transduction | |
Anti-NESTIN Antibody | MAB1259-SP | R&D | |
Anti-OLIG2 Antibody | MABN50 | Millipore | |
Anti-PAX6 Antibody | PRB-278P | Covance | |
Anti-SOX2 Antibody | MAB2018 | R&D | |
Anti-TBR1 Antibody | AB2261 | Millipore | |
Anti-TBR2 Antibody | ab183991 | Abcam | |
Anti-TUJ1 Antibody | 801213 | Biolegend | |
Apo-transferrin | T1147 | Sigma | |
BrainPhys Neuronal Medium N2-A & SM1 Kit | 5793 – 500mL | Stem cell tecnhnologies | |
Chemically defined lipid concentrate | 11905031 | ThermoFisher | |
Coverslips 24x60mm | 631-1575 | VWR | |
Crystallization-purified BSA | 5470 | Sigma | |
DAPI | 10236276001 | Sigma | |
Dibutyryl cAMP | SC- 201567B -500mg | Frilabo | |
DMEM-F12 | 32500-035 | ThermoFisher | |
Fetal bovine serum | A3840001 | ThermoFisher | |
Gelatin from bovine skin | G9391 | Sigma | |
Glass Copling Jar | E94 | ThermoFisher | |
Glutamax I | 10566-016 | ThermoFisher | |
Glycine | MB014001 | NZYtech | |
Ham’s F12 | 21765029 | ThermoFisher | |
Human Episomal iPSC Line | A18945 | ThermoFisher | iPSC6.2 |
IMDM | 12440046 | ThermoFisher | |
Insulin | 91077C | Sigma | |
iPS DF6-9-9T.B | WiCell | ||
Iso-pentane | PHR1661-2ML | Sigma | |
L-Ascorbic acid | A-92902 | Sigma | |
Matrigel | 354230 | Corning | basement membrane matrix |
Monothioglycerol | M6154 | Sigma | |
Mowiol | 475904 | Millipore | mounting medium |
mTeSR1 | 85850 -500ml | Stem cell technologies | |
N2 supplement | 17502048 | ThermoFisher | |
Neurobasal | 12348017 | ThermoFisher | |
Paraformaldehyde | 158127 | Sigma | |
PBS-0.1 Single-Use Vessel | SKU: IA-0.1-D-001 | PBS Biotech | |
PBS-MINI MagDrive Base Unit | SKU: IA-UNI-B-501 | PBS Biotech | |
Recombinant human BDNF | 450-02 | Peprotech | |
Recombinant human bFGF/FGF2 | 100-18B | Peprotech | |
Recombinant human FGF19 | 100-32 | Peprotech | |
Recombinant human GDNF | 450-10 | Peprotech | |
Recombinant human SDF1 | 300-28A | Peprotech | |
ROCK inhibitor Y-27632 | 72302 | Stem cell technologies | |
SB431542 | S4317 | Sigma | |
Sucrose | S7903 | Sigma | |
SuperFrost Microscope slides | 12372098 | ThermoFisher | adhesion microscope slides |
Tissue-Tek O.C.T. Compound | 25608-930 | VWR | |
Tris-HCL 1M | T3038-1L | Sigma | |
Triton X-100 | 9002-93-1 | Sigma | |
Tween-20 | P1379 | Sigma | |
UltraPure 0.5M EDTA, pH 8.0 | 15575020 | ThermoFisher |