The authors thank la Ligue Genevoise Contre le Cancer (Geneva, Switzerland), ISREC Foundation (Lausanne, Switzerland), and the Clayton Foundation for Research (Houston, TX, USA) for financial support. Moreover, the authors thank HES-HO and the Wyss Center for financial support. We thank the Krause&#39;s lab for helpful discussions and support and Dr. Halah Kutaish for proofreading.

This protocol follows the guidelines of University of Geneva&#39;s human research ethics committee.
1. Maintenance and culture of undifferentiated human embryonic stem cells (hESCs)
<ol>
	<li>Perform maintenance and expansion of hSECs on feeder-free conditions by pre-coating dishes with a specific extracellular matrix.
	<ol>
		<li>Thaw 300 &#181;L of extracellular matrix at 4 &#176;C (typical range concentration 18-22 mg/mL, keep on ice) and gently mix with 15 mL of cold DMEM medium to avoid a premature gelation of the extracellular matrix. Add 7.5 mL of the extracellular matric to both T150 flasks.</li>
		<li>Incubate the dishes coated with extracellular matrix at 37 &#176;C for at least 1 h (maximum overnight).</li>
		<li>Remove the medium and seed hESCs to a density of 6.5 x 104 cell/cm2.</li>
	</ol>
	</li>
	<li>Maintain H1 (hESC cell line) in hESC medium and 1% penicillin/streptomycin.</li>
	<li>Pass the cells with enzymatic procedure: add 7.5 mL of enzymatic solution to a T75 cm2 flask over a period of 1-2 min at 37 &#176;C. Once cells are completely detached, add 7.5 mL of DMEM-F12 then centrifuge for 5 min at 300 x g. To allow for better survival, re-plate cells at the desired density onto extracellular matrix-coated dishes, in the same medium containing Rho-associated protein kinase (ROCK) inhibitor (10 &#181;M) for 24 h.</li>
</ol>
2. hESC-derived neural organoids for GBM studies
<ol>
	<li>24 h before starting the 3D culture, replace the hESC medium with a serum-free medium supplemented with 10 &#181;M ROCK inhibitor (both components are necessary to support cell survival and spontaneous neurosphere formation during the aggregation phase in a microwell plate). The cells should be at 60% confluency. The next day (day 0), detach hESC colonies as single cells: remove the medium, then rinse with PBS without Ca2+/Mg2+, add 5 mL of enzymatic dissolution solution, and incubate at 37 &#176;C for 1-2 min.</li>
	<li>Collect the cells in serum-free medium with 10 &#181;M of ROCK inhibitor and centrifuge the cells at 300 x g for 5 min. Remove the supernatant and count the cells in 10 mL of serum-free medium supplemented with 10 &#181;M of ROCK inhibitor.</li>
	<li>In parallel, rinse the microwell plate with 2 mL of serum-free medium per well and centrifuge the plate at 1200 x g for 5 min to remove all bubbles, which can prevent neurosphere formation.</li>
	<li>Prepare 28.2 x 106 of cells in 12.5 mL of serum-free medium supplemented with 10 &#181;M ROCK inhibitor. Dispense 1000 cells/microwell. Centrifuge the cells at 300 x g for 5 min and place the plate in the incubator at 37 &#176;C overnight (maximum 36 h). For example: to obtain 30 human neural organoids, use one T150 flask at 70%-80% of confluence (about 30 million cells).</li>
	<li>The next day (day 1), collect the spheres (with a P1000) and place them in a 6 well plate. In each well, add 2 mL of B27 medium and DMEM-F12 GlutaMAX and Neurobasal medium (mix at 1:1), supplemented with 1% B27 supplements and 1% non-essential amino acids (NEAA). To promote fast neural induction, supplement the medium with dual-SMAD inhibition cocktail, composed of 10 &#181;M TGF&#946;/Activin/Nodal inhibitor and 0.5 &#181;M bone morphogenic protein (BMP) inhibitor. From this step forward, the spheres are cultured in rotation (60 rpm, orbital shaker). The rotation is critical to prevent the spheres from sticking together or to the plate.</li>
	<li>Change the medium every 2-3 days: bend the plate and let the spheres fall down for 5 min, remove half of the medium (2 mL), and add 2 mL of fresh B27 medium supplemented with growth factors and inhibitors. Do not centrifuge the spheres.</li>
	<li>Perform neural induction according to the following time course:
	<ol>
		<li>From days 1-4, culture the spheres in B27 medium supplemented with dual-SMAD. The dual-SMAD inhibition cocktail (10 &#181;M TGF&#946;/Activin/Nodal inhibitor and 0.5 &#181;M BMP inhibitor) promote the neural induction.</li>
		<li>From days 4-11, promote proliferation of hESC-derived neural rosettes (into the spheres), by adding 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast factor (bFGF) to the B27 medium supplemented with dual-SMAD cocktail. 
		NOTE: On day 11, most of the cells should be positive for Nestin.</li>
		<li>From days 11-13, culture the spheres in B27 medium supplemented with 0.5 &#181;M BMP inhibitor.</li>
		<li>From days 13-21, culture the spheres in B27 medium supplemented with 10 ng/mL glial derived neurotrophic factor (GDNF), 10 ng/mL brain derived neurotrophic factor (BDNF) and 1 &#181;M of &#947;-secretase inhibitor. GDNF and BDNF promote neuronal and glial differentiation. The &#947;-secretase inhibitor allows for greater neural maturation.</li>
		<li>On day 21, plate the spheres (about 1,000 spheres) on a hydrophilic polytetrafluoroethylene (PTFE) membrane (6 mm diameter, 0.4 &#181;m) deposited on a culture plate insert designed for 6 well plate. Stop any rotation from this step. The presence of rosettes, observed with a bright-field microscope, indicate the initiation of neural differentiation. The neural rosettes can be observed 2-3 days after plating spheres on the PTFE membrane.</li>
		<li>Add 1 mL of B27 medium supplemented with growth factors and inhibitors (as followed) to each well underneath the membrane insert, every 2-3 days (usually on Monday, Wednesday and Friday), for a following 3 weeks of differentiation.</li>
		<li>From days 21-25, cultivate human neural organoids in the same neural maturation medium (Cf. step 2.7.4).</li>
		<li>From days 25-28, only complement B27 medium with 1 &#181;M &#947;-secretase inhibitor.</li>
		<li>From days 28-39, stop adding the &#947;-secretase inhibitor and continue human neural organoid culture in B27 medium only. 
		NOTE: After 3 weeks, neural organoids are ready to use for GIC implantation. Along the neural maturation, a decrease of neural immature marker Nestin and increase of mature neural markers &#946;3-tubulin and GFAP were observed.</li>
	</ol>
	</li>
</ol>
3. Isolation and cultivation of glioblastoma-initiating cells (GICs)
<ol>
	<li>Isolate GICs by fragmenting a high grade human GBM biopsy. Transfer the piece of tumor in a beaker containing 0.25% trypsin in 0.1 mM EDTA (4:1) and slowly stir at 37 &#176;C for 30-60 min (depending on tumor size).</li>
	<li>Plate the dissociated cells in 75 cm2 tissue culture flasks plated at 2,500-5,000 cells per cm2 in GIC medium: DMEM/F-12 medium (1:1) containing 1% N2, 1% B27, and 1% G5 supplements (to favor GIC survival), supplemented with bFGF and EGF (both at 10 ng/Ml, to promote stemness) and 1% of penicillin/streptomycin.</li>
	<li>Once the GIC is well-established and growing, remove the N2 and G5 supplements from the GIC medium.</li>
	<li>One day before adding the cells onto the organoid, dissociate the GICs and count them.</li>
	<li>Rinse the microwell plate with 2 mL of GIC medium and centrifuge the plate at the maximum speed to remove bubbles (1000 x g). Place the GICs at 1,000 cells to obtain one gliomasphere per microwell. Incubate overnight at 37 &#176;C (Figure 1C). This step is key and allows for well-calibrated GICs (an example of necrotic and over-sized GICs is shown in Figure 2A,C).</li>
	<li>To initiate GBM invasion, add one gliomasphere on top of the neural tissue with a large bore pipet tip (Figure 1F). Carefully place the 6 well plate back in the incubator.</li>
</ol>
4. hESC-derived dopaminergic organoids for PD studies
<ol>
	<li>Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then replace stem cell media used to maintain pluripotency features of hESCs with a serum-free medium. Start neural induction by supplementing culture medium with 0.5 &#181;M BMP inhibitor and 10 &#181;M TGF&#946;/Activin/Nodal inhibitor (dual-SMAD inhibition cocktail), then add 10 &#181;M ROCK inhibitor for 24 h to increase the survival rate of cells during passage.</li>
	<li>Day 1: Prepare the microwell plate with 2.5 mL per well of serum-free medium supplemented with 0.5 &#181;M BMP inhibitor, 10 &#181;M TGF&#946;/Activin/Nodal inhibitor, and 10 &#181;M ROCK inhibitor. To specify cells towards the ventral pattern of the neural tube, add 100 ng/mL Sonic Hedgehog (SHH), 100 ng/mL fibroblast growth factor 8 (FGF8), and 2 &#181;M smoothened agonist. Centrifuge the plate (only with medium and without cells) at 1200 x g for 5 min to remove air bubbles from the microwells.
	<ol>
		<li>After 1 day of neural induction in 2D, remove the medium and quickly wash with PBS without Ca2+/MgCl2+. Dissociate the colonies in single cells suspension by adding 7.5 mL of recombinant enzymatic solution in a T75 cm2 flask. Incubate for 2 min at 37 &#176;C then complete with 7.5 mL of DMEM-F12.</li>
		<li>Collect the cell suspension and centrifuge at 300 x g for 5 min. Remove the supernatant and count the cells in the same medium used to prepare the microwell plate.</li>
		<li>Adjust the medium volume to obtain a cell suspension allowing to form neurospheres containing 1000 cells per microwell (for example, the microwell plate used here contains 4,700 microwells per well). So, prepare 4.7 million cells in 2.5 mL of medium and add it to the previous 2.5 mL of medium already placed in the plate.</li>
		<li>In order to correctly distribute the cells in each microwell, gently shake the plate, and centrifuge the microwell plate 300 x g for 5 min. Incubate the plate at 37 &#176;C for 24 h to generate spheres.</li>
	</ol>
	</li>
	<li>Day 2: Gently flush the microwells with the medium and collect then transfer the spheres in tissue-treated six-well plate. Replace medium with Neurobasal medium supplemented with 1% B27, 1x NEAA, 2 mM L-glutamine, and 1% of penicillin/ streptomycin. Additionally, add regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition small molecules.
	<ol>
		<li>Place spheres in rotation at 37 &#176;C (60 rpm, orbital shaker) and change half-medium freshly supplemented every 2-3 days.</li>
	</ol>
	</li>
	<li>Day 3: To enhance neural induction and convert to neural progenitors with a midbrain identity, supplement the medium with 3 &#181;M GSK-3&#946; inhibitor, which activates the Wnt/&#946;-catenin pathway. Maintain GSK-3&#946; inhibitor in the medium up to day 13. Split into two new tissue-treated 6 well plates to reduce both sphere density per well and avoid sphere aggregation. 
	NOTE: At Day 8, most of the cells should be positive for Nestin.</li>
	<li>Day 8: Start the neural maturation: replace regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition cocktail with 0.5 mM dibutyryl cAMP (to favor maturation), 20 nM inhibitor of histone deacetylase (for cell cycle exit), 1 &#181;M &#947;-secretase inhibitor and growth factors, 10 ng/mL GDNF, 10 ng/mL BDNF, 1 ng/mL transforming growth factor &#946;3 (TGF&#946;3), and 5 ng/mL FGF20 (both favor DA progenitor survival). Change the medium every 2-3 days.</li>
	<li>Day 21: Generate the neural organoid: seed around 100 neurospheres under air-liquid interface conditions on PTFE membrane (6 mm diameter). Transfer the membrane to a culture plate insert (0.4 mm) and add 1.2 mL of neural maturation medium used for neurosphere differentiation as previously described.
	<ol>
		<li>Stop any rotation from this step. Change the medium every 2-3 days until the required differentiation time point is achieved. 
		NOTE: Regarding neural maturation, a decrease of the neural immature marker Nestin and increase of mature neural markers &#946;3-tubulin and GFAP were observed. High TH and NURR1 expressions were observed (Figure 3C) and confirm neural organoid maturity11.</li>
	</ol>
	</li>
</ol>
5. Quantification of TH and Nurr1 gene expression for validation of dopaminergic differentiation
<ol>
	<li>RNA extraction: On the indicated day of differentiation, lyse 40 neurospheres with 350 &#181;L of RLT buffer (provided in RNA extraction kit) supplemented with 3.5 &#181;L of 2-mercaptoethanol. Extract the RNA from the lysed neurospheres using an RNA extraction kit following the manufacturer&#39;s instructions.</li>
	<li>Quantify total RNA concentrations.</li>
	<li>Perform reverse transcription of 300 ng of the total RNA extraction using reverse transcription kit for quantitative real-time polymerase chain reaction (qPCR) and follow the manufacturer&#39;s instructions .</li>
	<li>Perform qPCR analysis on real-time PCR detection systems, based on asymmetrical cyanine dye detection. Normalize the data with housekeeping genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1-alpha (EF1). Sequences of primers are described in Table 1.</li>
</ol>
6. High pressure liquid chromatography (HPLC) detection
<ol>
	<li>Use high pressure liquid chromatography (HPLC) with electrochemical detection to detect the presence of dopamine. Dopamine was extracted by lysing neural organoids in 100 mL of 0.1 N perchloric acid (HClO4) for 15 min at 4 &#176;C with a vigorous vortexing every 5 min. After centrifugation, collect and store the supernatant at -20 &#176;C for dopamine dosage.</li>
	<li>Use a C-18 column (5 &#181;m, 4.6 mm x 150 mm) to separate the analytes by reversed-phase HPLC in isocratic mode at a flow rate of 1 mL/minute. Detection of dopamine should be carried out using a coulometric detector with the conditioning cell set at a potential of +200 mV.</li>
</ol>
7. Raw data recording with microelectode array (MEA) platform
<ol>
	<li>Use a dissection microscope to transfer neurospheres to the center of a porous MEA device.</li>
	<li>Use an amplifier and data acquisition system for electrophysiological recordings. Measure the signal-to-noise ratio (SNR) as the standard deviation of the voltage during a 5 min recording, using the signal as the average peak-to-peak voltage of the spikes recorded in the same 5 min periods.</li>
</ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2016+World+Health+Organization+Classification+of+Tumors+of+the+Central+Nervous+System:+a+summary.">The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary.</a> Acta Neuropathologica. 131 (6), 803-820 (2016).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> New England Journal of Medicine. 352 (10), 987-996 (2005).</li><li>Daphu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+animal+models+for+studying+brain+metastasis:+value+and+limitations.">In vivo animal models for studying brain metastasis: value and limitations.</a> Clinical and Experimental Metastasis. 30 (5), 695-710 (2013).</li><li>Huszthy, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+primary+brain+tumors:+pitfalls+and+perspectives.">In vivo models of primary brain tumors: pitfalls and perspectives.</a> Neuro Oncology. 14 (8), 979-993 (2012).</li><li>Nayernia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+brain+tumor+cell+invasion+of+engineered+neural+tissues+and+in+vivo+features+of+glioblastoma.">The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma.</a> Biomaterials. 34 (33), 8279-8290 (2013).</li><li>Cosset, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tissue+engineering+allows+the+identification+of+active+miRNA+regulators+of+glioblastoma+aggressiveness.">Human tissue engineering allows the identification of active miRNA regulators of glioblastoma aggressiveness.</a> Biomaterials. 107, 74-87 (2016).</li><li>Pringsheim, T., Jette, N., Frolkis, A., Steeves, T. 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Clinical, morphological and neurochemical correlations.</a> Journal of Neurological Science. 20 (4), 415-455 (1973).</li><li>Tieng, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+midbrain+organoids+containing+long-lived+dopaminergic+neurons.">Engineering of midbrain organoids containing long-lived dopaminergic neurons.</a> Stem Cells and Development. 23 (13), 1535-1547 (2014).</li><li>Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Biomimetic+Cultures:+The+Next+Platform+for+Cell+Biology.">3D Biomimetic Cultures: The Next Platform for Cell Biology.</a> Trends in Cell Biology. 26 (11), 798-800 (2016).</li><li>Friedl, P., Sahai, E., Weiss, S., Yamada, K. 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The authors declare no competing financial interests.

One of the most critical aspects of this protocol includes the maintenance of hESC pluripotency during cell culturing and close monitoring of the spheres and neural organoid morphology. hESCs are very sensitive, and every manipulation can lead to early uncontrolled differentiation as well as cell death. In order to increase experimental reproducibility and avoid the occurrence of abnormal karyotype events, it is advised to cryopreserve several batches of hESCs at the lowest passage after validation of their chromosome stability. Moreover, it is recommended to thaw a new vial for each experiment and check the behavior of the cells every day. If the spheres are less refractive with abnormal higher size, they will likely start to aggregate and die.
One improvement upon this system is either perfusion or implementing a vascularized system (by adding endothelial cells or within a 3D fluidic microchip)12,13. However, controlling the thickness of the neural organoid (&#8804;300 &#181;m) allows efficient passive perfusion of oxygen and nutriments and prevents necrosis. Another improvement is the introduction of immune cells (microglia). With these limitations in mind, neural organoids plus a GIC system may be a relevant tool for several reasons. First, this system allows drug screening to monitor how a therapeutic compound may affect an organoid or tumor cell. Second, cell-to-cell interactions can be studied, and micro-environmental determinants underlying individual and collective invasions can be visualized and explored5,6,13.
In the context of Parkinson&#39;s disease, a neural organoid enriched in DA neurons can represent a relevant and accurate 3D model to study disease development. In previous studies, Parkinson&#39;s patient-derived induced pluripotent stem cells differentiated towards DA neurons have been used to study the affected neuronal subtypes. Of note, some disease-related phenotypes such as the accumulation of &#945;-synuclein and sensitivity to oxidative stress have been observed14,15. Moreover, the neural organoid may be used as a tool to screen therapeutic molecules. However, specific and relevant readouts should be set up to evaluate DA neuron survival and functionality, such as dopamine production and electrophysiological activity. Altogether, this protocol provides two standardized and accurate stem cell-based approaches to generate neural organoids.

The World Health Organization (WHO) classifies astrocytomas as low grade (grade I to II) or high grade (grade III and IV). Glioblastoma multiforme (GBM) is an astrocytoma grade IV, the most lethal of primary brain tumors, that is resistant to all current forms of treatments1. Despite standard-of-care therapy including neurosurgery, chemotherapy, and radiotherapy, GBM remains fatal and the 15-month overall survival rate has not dramatically changed over the past 15 years2. To make significant progress in understanding GBM pathogenesis, the use of relevant models is key. So far, the study of GBM has relied on cell lines, rodent organotypic slices, and xenotransplantation of patient-derived cells into mice or transgenic mice developing spontaneous tumors3,4. Although these models have been useful to study brain metastasis and tumor aggressiveness, they are restricted by differences among species, and resulting conclusions may be incorrectly translated to human tissues. Moreover, existing models with human cells are also limited by the absence of host tissue/tumor interactions3,4. Experimental models are critical for the translation from basic science to therapeutic targets. Therefore, describing a protocol to produce in vitro human neural organoids co-cultured with GBM-initiating cells (GICs) can provide a relevant system that mimics morphological and functional features of GBM development. This system reproduces some in vivo&#160;features of GBM developmentsuch as diffuse migration of invading cells and necrosis areas, and it highlights gene expression relevant to tumor biology. As previously revealed, some critical microRNAs are induced during GIC development within 3D nervous tissue5,6.
PD is a major neurodegenerative disorder and associated with the degeneration of multiple neuronal subtypes7. Even if a progressive onset of symptoms (e.g., bradykinesia, asymmetric rest tremor, rigidity and posture instability) characterizes the disease, its exact etiology is not clearly established. Indeed, many studies have highlighted evidence that major risk factors can result from a combination of genetic and environmental factors. Parkinsonian symptoms are associated with the bilateral degeneration of dopaminergic neurons in the substancia nigra (SN), leading to the disappearance of dopaminergic (DA) axons projecting to the striatum8,9. Therefore, the reduction of striatal dopamine levels is correlated with progression of motor dysfunction in PD patients. Dopaminergic neurons contain tyrosine hydroxylase (TH), a key enzyme in the synthesis of catecholaminergic neurotransmitters that converts the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA, a dopamine precursor) to dopamine10. Early loss of TH activity followed by a decline in TH protein expression is a hallmark of PD.
This study describes two protocols using human neural organoids, with one specifically oriented towards a midbrain-like phenotype enriched with TH-positive cells.

<table><tbody><tr><td>6-well plate (6-well plate)</td><td>Falcon / Corning</td><td>07-201-588</td><td></td></tr><tr><td>ABI Prism 7900 HT detection system&amp;nbsp; (Real-Time PCR detection systems)</td><td>Applied Biosystems</td><td>Discontinued</td><td></td></tr><tr><td>Aggrewell 400 (Microwell culture plates )</td><td>StemCell Technologies</td><td>34421</td><td></td></tr><tr><td>Amplifier (W2100-HS32)&amp;nbsp; (Amplifier)</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>Anti-EGFR (phospho Y1101) antibody</td><td>Abcam</td><td>ab76195</td><td>1/100 dilution</td></tr><tr><td>Anti-GFAP Antibody</td><td>Dako&amp;nbsp;</td><td>Z334&amp;nbsp;</td><td>1/1000 dilution</td></tr><tr><td>Anti-Nestin, Human Antibody</td><td>Millipore&amp;nbsp;</td><td>ABD69&amp;nbsp;</td><td>1/400 dilution</td></tr><tr><td>Anti-Synapsin I Antibody</td><td>Chemicon&amp;nbsp;</td><td>AB1543P&amp;nbsp;</td><td>1/500 dilution</td></tr><tr><td>B27 supplements (B27)</td><td>Life Technologies / Invitrogen</td><td>1238</td><td>For both protocol, stock solution 100x, final solution 1x</td></tr><tr><td>Brain-derived neurotrophic factor&amp;nbsp; (BDNF)</td><td>Cell Guidance</td><td>GFH1-2</td><td>For both protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 20 ng/mL</td></tr><tr><td>CHIR-99021 (GSK-3&amp;beta; inhibitor )</td><td>Axon Medchem</td><td>ct99021</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 7.5 mM in DMSO, final solution 3 &amp;micro;M</td></tr><tr><td>Compound E a &amp;gamma;&amp;ndash;secretase inhibitor (&amp;gamma;&amp;ndash;secretase inhibitor)</td><td>Calbiochem</td><td>CAS 209986-17-4</td><td>For both protocol (gamma-secretase inhibitor XXI),&amp;nbsp; stock solution 5 mM in DMSO, final solution 1 &amp;micro;M</td></tr><tr><td>Coulochem III&amp;nbsp; (Coulometric detector parameters)</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Dibutyryl cyclic-AMP (Dibutyryl cAMP)</td><td>Sigma</td><td>D0627</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 0.5 M in DMSO, final solution 0.5 mM</td></tr><tr><td>Dimethyl Sulfoxide Pure (DMSO)</td><td>Sigma-Aldrich</td><td>C6164</td><td>Compounds solvent, ready to use</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>12491-015</td><td>For cell culture, ready to use</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium Mixture F-12 (DMEM-F12)</td><td>Gibco</td><td>11320033</td><td>For cell culture, ready to use</td></tr><tr><td>EDTA 0.1 mM (EDTA)</td><td>Life Technologies</td><td>AM9912</td><td>For cell culture, ready to use</td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Gibco</td><td>PHG0313</td><td>For GIC culture,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 10 ng/mL</td></tr><tr><td>Fibroblast Growth Factor 20 (FGF20)</td><td>Peprotech</td><td>100-41</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 5 ng/mL</td></tr><tr><td>fibroblast growth factor 8&amp;nbsp; (FGF8)</td><td>Peprotech</td><td>GFH176-5</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 100 ng/mL</td></tr><tr><td>Fibroblast growth factor-basic (bFGF)</td><td>Gibco</td><td>PHG0024</td><td>For GIC culture,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 10 ng/mL</td></tr><tr><td>G5 supplements (G5)</td><td>Invitrogen</td><td>17503012</td><td>For GIC culture, stock solution 100x, final solution 1x</td></tr><tr><td>Glial cell-derived neurotrophic factor (GDNF)</td><td>Cell Guidance</td><td>GFH2-2</td><td>For both protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 20 ng/mL</td></tr><tr><td>Hydrophilic polytetrafluoroethylene&amp;nbsp; membrane (PTFE membrane)</td><td>BioCell-Interface</td><td>Discontinued</td><td></td></tr><tr><td>LDN-193189 (BMP inhibitor)</td><td>Axon Medchem /Stemgen</td><td>04-0072-02 /1509</td><td>Dual/Smad,&amp;nbsp; stock solution 5 mM in DMSO, final solution 0.5 &amp;micro;M</td></tr><tr><td>L-glutamine (L-glutamine)</td><td>Gibco</td><td>25030081</td><td>L-Glutamine (200 mM), stock solution 200 mM, final solution 2 mM</td></tr><tr><td>Matrigel (extracellular matrix)</td><td>BD Biosciences</td><td>354277</td><td>hESC-qualified Matrix, stock solution 18-22 mg/mL, final solution 180-220 &amp;micro;g/mL</td></tr><tr><td>Millicell-CM Culture plate insert&amp;nbsp; (0.4 &amp;micro;m) (Culture plate insert)</td><td>Millipore</td><td>PICM03050</td><td></td></tr><tr><td>Monoclonal Anti-&amp;beta;-Tubulin III antibody</td><td>Sigma&amp;nbsp;</td><td>T8660&amp;nbsp;</td><td>1/1000 dilution</td></tr><tr><td>MS Orbital Shaker, MS-NOR-30 (Orbital shaker)</td><td>Major Science</td><td>MS-NRC-30</td><td></td></tr><tr><td>N2 supplements (N2)</td><td>Invitrogen</td><td>17502-048</td><td>For GIC culture, stock solution 100x, final solution 1x</td></tr><tr><td>Nanodrop (Nanodrop)</td><td>Thermo Fisher Scientific</td><td>Discontinued</td><td></td></tr><tr><td>Neurobasal (Neurobasal)</td><td>Life Technologies / Gibco</td><td>21103049</td><td>Maintenance and maturation embryonic neuronal cell populations&amp;nbsp;, ready to use</td></tr><tr><td>Non-Essential Amino Acids&amp;nbsp; (NEAA)</td><td>Gibco</td><td>11140</td><td>Non-essential Amino Acids 100X, stock solution 100x, final solution 1x</td></tr><tr><td>Nurr1 Antibody (M-196)</td><td>Santa Cruz</td><td>Sc-5568</td><td>1/100 dilution</td></tr><tr><td>Nutristem&amp;nbsp; (hESC medium )</td><td>Biological Industries</td><td>05-100-1A</td><td>Stem cell media, ready to use</td></tr><tr><td>Penicilin / Streptomycin&amp;nbsp; (Penicilin / Streptomycin )</td><td>Life Technologies / Gibco</td><td>15140122</td><td>For cell culture, stock solution 5 mg/mL, final solution 50 &amp;micro;g/mL&amp;nbsp;</td></tr><tr><td>Perchloric acid 0.1N (HCLO4)</td><td>Merck</td><td>100519</td><td>For HPLC, ready to use</td></tr><tr><td>Phosphate Buffered Saline without Ca2+/Mg2+&amp;nbsp; (PBS without Ca2+/Mg2+ )</td><td>Life Technologies</td><td>14190250</td><td>For cell culture, ready to use</td></tr><tr><td>PrimeScript RT-PCR Kit (Reverse transcription kit)</td><td>Takara</td><td>RR014A</td><td></td></tr><tr><td>Purmorphamin (smoothened agonist)</td><td>Calbiochem</td><td>SML0868</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 10 mM in DMSO, final solution 2 &amp;micro;M</td></tr><tr><td>Rho-associated Kinase Y-27632 (ROCK)</td><td>Abcam Biochemicals</td><td>ab120129-1</td><td>Rock Inhibitor,&amp;nbsp; stock solution 50 mM in DMSO, final solution 10 &amp;micro;M</td></tr><tr><td>RNeasy mini kit (RNA extraction kit )</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>SB-431542 (TGF&amp;beta;/Activin/Nodal inhibitor )</td><td>Ascent</td><td>Asc- 163</td><td>Dual-Smad,&amp;nbsp; stock solution 50 mM in DMSO, final solution 10 &amp;micro;M</td></tr><tr><td>Sonic Hedgehog (SHH)</td><td>Cell Guidance</td><td>GFH168-5</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure H2O, final solution 100 ng/mL</td></tr><tr><td>StemPro Accutase (hESC enzymatic solution)</td><td>Gibco</td><td>A11105-01</td><td>hESC enzymatic solution, ready to use</td></tr><tr><td>Symmetry C-18,5 mm (4.6 150mm2)&amp;nbsp; (Reversed-phase column)</td><td>Waters Corporation</td><td></td><td></td></tr><tr><td>T150 flask (T150 flask)</td><td>Falcon</td><td>08-772-1F</td><td></td></tr><tr><td>TH Antibody (F-11)</td><td>Santa Cruz</td><td>Sc-25269</td><td>1/200 dilution</td></tr><tr><td>Transforming Growth Factors beta 3 (TGF&amp;beta;3)</td><td>Cell Guidance</td><td>GFH109-2</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 100 &amp;micro;g/mL in pure ethanol , final solution 1 ng/mL</td></tr><tr><td>Trichostatine A (inhibitor of histone deacetylase )</td><td>Sigma</td><td>T8552</td><td>For Dopaminergic protocol,&amp;nbsp; stock solution 100 &amp;micro;M in DMSO, final solution 20 nM</td></tr><tr><td>TrypLE (recombinant enzymatic solution)</td><td>Invitrogen</td><td>12604021</td><td>recombinant enzymatic solution, ready to use</td></tr><tr><td>Trypsin 0.25% (enzymatic solution)</td><td>Life Technologies</td><td>15050065</td><td>enzymatic solution, ready to use</td></tr><tr><td>W2100, Multi Channel Systems (Data acquisition system )</td><td></td><td>WAT045905</td><td></td></tr><tr><td>X-vivo (serum free medium)</td><td>Lonza</td><td>BE04-743Q</td><td>serum free medium, ready to use</td></tr></tbody></table>

human neural organoids for studying brain cancer and neurodegenerative diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

The critical steps of this protocol must be well-identified and handled properly. Therefore, a diagram of culture conditions indicating the time-lapse for each step as well as the compounds used for the differentiation protocol are illustrated in Figure 1A and Figure 3A for NO plus GBM and DA neural organoids, respectively. Figure 1B,C,D,E,F illustrates the cells, spheres, and NO and show the typical morphology for each step. Figure 1G,H,I illustrates immunofluorescence staining with some neural markers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59682/59682fig1.jpg" /> 
Figure 1: Human neural organoid (NO)&#160;differentiation protocol. (A) Standardized protocol for the generation of NO derived from human embryonic stem cells (hESC). (B) hESCs are maintained on extracellular matrix in hESC medium. (C) Microwell plates were used to generate calibrated neurospheres. At 2 weeks, neurospheres were plated onto the insert containing a PTFE membrane (scale bar = 50 &#181;m). (D) Macroscopic view of NO into the insert in one well of a 6 well plate. During the first days, rosettes were observed (black arrow) (E). (F) Macroscopic view of a NO plus GIC sphere on the top. (G-I) Immunofluorescence analysis of NO plus GIC sphere (EGFR-positive; scale bar = 50 &#181;m) (G) and NO alone, which showed immune reactivity for the neuronal marker &#946;III-tubulin and slightly positive for nestin; however, synapsin 1 showed a weak signal (H,I) (scale bars = 100 &#181;m and 50 &#181;m, respectively). <a href="https://www.jove.com/files/ftp_upload/59682/59682fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59682/59682fig2.jpg" /> 
Figure 2: Illustration of necrotic spheres and immature NO. The neurospheres (A) and NO (B) can undergo necrosis when they are too numerous in the well or oversized (C) (scale bar = 10 &#181;m). (D) One GIC infected with a tomato reporter help to track tumor cell invasion in NO, scale bar, 10 &#181;m. Example of immature NO with neural tubes (E) and no neural tubes (F) (scale bar = 50 &#181;m). <a href="https://www.jove.com/files/ftp_upload/59682/59682fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59682/59682fig3.jpg" /> 
Figure 3: Standardized protocol for generation of DA neural organoid&#160;and electrophysiological and morphological analysis. (A) Standardized protocol for the generation of DA neural organoids. (B) Immunofluorescence analysis of DA neural organoid; TH-immunoreactive cells co-expressing Nurr1, a midbrain specific marker (scale bar = 50 &#181;m). Data are represented as mean &#177; SEM (n = 3). (C) Graphs represent kinetics of TH and Nurr1 gene expression evaluated by qRT-PCR. (D) Representative HPLC: dopamine peak (arrow) was detected by HPLC from DA neural organoid lysate. (E) Example of raw data recorded with MEA platform. Each spike is displayed by a vertical line (time stamps), whereas the remaining trace is noise. (F) Picture representing a neurosphere deposited on the MEA. (G) Superposition of typical spikes (blue and red curves) detected from the raw data. The black bold curve indicates the average of the corresponding red curves. (H) Raster plot showing the time stamps associated with each spike detected. The different colors highlight the different electrodes. <a href="https://www.jove.com/files/ftp_upload/59682/59682fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gene</td>
			<td>Foward</td>
			<td>Reverse</td>
		</tr>
		<tr>
			<td>Nurr1</td>
			<td>GGCTGAAGCCATGCCTTGT</td>
			<td>GTGAGGTCCATGCTAAACTTGACA</td>
		</tr>
		<tr>
			<td>TH</td>
			<td>GCACCTTCGCGCAGTTCT</td>
			<td>CCCGAACTCCACCGTGAA</td>
		</tr>
		<tr>
			<td>EEF1</td>
			<td>AGCAAAAATGACCCACCAATG</td>
			<td>GCCTGGATGGTTCAGGATA</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>GCACAAGAGGAAAGAGAGAGAACC</td>
			<td>AGGGGAGATTCAGTGTGGGT</td>
		</tr>
	</tbody>
</table>
Table 1: Primers used in this protocol.

Watch this Scientific Journal Video about Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases at JoVE.com

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

human-neural-organoids-for-studying-brain-cancer-neurodegenerative

Research

JoVE Journal

Bioengineering

9.8K Views.  University of Geneva. In de regeneratieve geneeskunde is een robuust protocol van differentiatie van pluripotente stamcellen naar een specifiek neuraal celtype een waardevol hulpmiddel. In ons geval waren deze protocollen zeer nuttig om zowel de ontwikkeling van glioblastoom als de ziekte van Parkinson te bestuderen. Ten eerste heeft de generatie van driedimensionale organoïde het voordeel van het nauw nabootsen van fysiologische ontwikkeling, en ook de productie van grootte gekalibreerde neurosferen uit pluripot...

Video: Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Developmental Biology

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

Cancer Research

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Neuroscience

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

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