We thank Valeria Fernandez-Vallone for the original instructions for the Miltenyi Neural Dissociation kit. We also thank the Genomics Technology Platform of the Max Delbrueck Centrum for providing the recipe for the NP40 lysis buffer and valuable advice setting up this protocol. We also thank Margareta Herzog and Alexandra Tschernycheff for the lab organizational support.

The described protocol is performed in a biosafety level 1 laboratory of the Max Delbr&#252;ck Center for Molecular Medicine (approval number: 138/08), in accordance with the requirements and in compliance with EU and national rules on ethics in research.
1. Derivation of forebrain organoids from induced pluripotent stem cells (iPSCs)
NOTE: This protocol was tested for several different iPSC lines cultured in a variety of stem cell media from different companies (Table 1). The generation of forebrain organoids is highly dependent on high quality iPSCs and a confluence of 60%-70% prior to starting the protocol. Here we used a commercially available cell line (see Table of Materials).
<ol>
	<li>Day 0: Embryoid body formation
	<ol>
		<li>For treatment of 96-well plate with pluronic solution, add 80 &#181;L of sterile filtered 2% pluronic solution per well of a 96-well U-bottom plate. Incubate for at least 15 min at room temperature (RT) or 37 &#176;C. 
		NOTE: Pluronic treated plates can be stored at 4 &#176;C for up to 2 weeks and can be used directly without any washing steps for embryoid body formation.</li>
		<li>Remove pluronic solution from each well using an aspirator or a multichannel pipette before seeding cells.</li>
		<li>Before starting, prepare the following reagents for one 96-well plate: 15 mL centrifuge tube with 5 mL of pre-warmed DMEM: F12 medium; 11 mL of stem cell medium supplemented with 50 &#181;M Y-27632, pluronic-treated plate (as described above).</li>
		<li>Aspirate media from one well of a 6-well plate of 60-70% confluent iPSCs. Wash cells once with PBS. Add 500 &#181;L of trypsin-based reagent and incubate for 2-6 min at 37 &#176;C. 
		NOTE: Incubation time for proper detachment of cells with trypsin-based reagent depends on cell density and the cell matrix-adhesive properties of each iPSC line. To avoid over digestion, it is advised to examine cell morphology after 2 min of incubation with trypsin-based reagent using a bright field microscope.</li>
		<li>Detach cells using a 1000 &#181;L pipette and transfer into the previously prepared 15 mL centrifuge tube with DMEM:F12 medium to stop dissociation.</li>
		<li>Centrifuge cell suspension for 3 min at 300 x g. Aspirate supernatant and keep the cell pellet. Resuspend the cells in 1 mL of stem cell medium supplemented with 50 &#181;M Y-27632.</li>
		<li>Count cells using a cell counter and add desired number of cells to the stem cell media supplemented with 50 &#181;M Y-27632. The generation of a single embryoid body requires 3,000-12,000 cells depending on the cell line.</li>
		<li>Transfer the cell suspension to a multichannel reagent reservoir using a 10 mL pipette. Using a multichannel pipette, plate 100 &#181;L/well of the cell suspension into the previously pluronic-treated 96-well plate.</li>
		<li>Place 96-well plate in an incubator at 37 &#176;C, 5% O2 and 5% CO2. To avoid any disturbance during embryoid body formation, refrain from moving the plate.</li>
	</ol>
	</li>
	<li>Day 2: Embryoid body formation examination
	<ol>
		<li>Examine embryoid body formation using a 10x objective of a bright field microscope. Embryoid bodies should display clear edges and minimal cell death.</li>
		<li>Aspirate as much medium as possible and replace with 100 &#181;L/well of stem cell medium. (Critical) Embryoid bodies are easily removed from the well during medium exchange. Pay attention to not remove them, therefore monitor medium after aspiration.</li>
	</ol>
	</li>
	<li>Day 3: Neuroectoderm induction
	<ol>
		<li>Aspirate as much medium as possible and replace with 120 &#181;L/well of induction medium and place plate at 37 &#176;C, 20% O2 and 5% CO2.</li>
	</ol>
	</li>
	<li>Day 6: Embedding
	<ol>
		<li>Depending on the iPSC line, 3 or 4 days are required to induce neuroectoderm emergence. Monitor embryoid bodies regularly. Once the edges turn bright, embryoid bodies are ready for embedding. Use one of the two different methods that can be used for embryoid body embedding as described below24.</li>
		<li>Embedding method 1: Cookie embedding
		<ol>
			<li>Thaw an aliquot of undiluted extracellular matrix gel on ice for 1-2 h. (Critical) Always keep extracellular matrix gel on ice to prevent it from solidifying. Prepare aliquots in advance to minimize thawing time and repeated thawing cycles.</li>
			<li>Cut the tip of a 200 &#181;L pipette using claw nippers and collect 16-32 embryoid bodies in one 1.5 mL microcentrifuge tube.</li>
			<li>Transfer the embryoid bodies in 67 &#181;L of the media into a new 1.5 mL microcentrifuge tube. Add 100 &#181;L of extracellular matrix gel using a cut 200 &#181;L pipette tip and mix gently.</li>
			<li>Evenly distribute the embryoid bodies in a 6 cm dish and place the plate for 5-10 min to allow the extracellular matrix gel to solidify.</li>
			<li>Add about 4 mL of differentiation media and incubate at 37 &#176;C, 20% O2 and 5% CO2. On the next day, place the dish on an orbital shaker (84 rpm). Ensure that all embryoid bodies detach from the bottom. If not, use a cut pipette tip and media to gently detach them.</li>
		</ol>
		</li>
		<li>Embedding method 2: Liquid embedding
		<ol>
			<li>Thaw an aliquot of undiluted extracellular matrix gel on ice for 1-2 h and place differentiation media on ice. (Critical) Media must be ice-cold when adding extracellular matrix gel.</li>
			<li>Once the medium is cold, add extracellular matrix gel to the final concentration of 2%. Cut the tip of a 200 &#181;L pipette using claw nippers and transfer up to 24 embryoid bodies to one well of a 6-well plate.</li>
			<li>Remove all excess media and add about 4 mL of 2% extracellular matrix gel/differentiation media into one well of a 6-well plate. Immediately place the plate on the orbital shaker (84 rpm).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 9-20: Perform a full media exchange using differentiation media every 3-4 days.</li>
	<li>Day 21-30: Transfer organoids into maturation media and perform full media exchanges every 3-4 days.</li>
	<li>Dissociation of organoids: For single nuclei and cell dissociation, use organoids of high quality. To avoid excess amounts of cell death, regularly cut the organoids to prevent the build-up of a necrotic core and allow them to recover for 2 weeks before dissociating them for further analysis.</li>
</ol>
2. Derivation of single cell from organoids
NOTE: Single cell dissociation is performed using the Neural Tissue Dissociation Kit (Table 2), which uses mechanical and enzymatic dissociation. Here we describe a manual mechanical dissociation. As an alternative, a dissociation machine can be used.
<ol>
	<li>Prepare STOP solution and 0.04% BSA on ice. Prepare enzyme mix 1 and 2 and 0.04% BSA in PBS and place on ice.</li>
	<li>Collect up to 5 organoids in one well of a 6-well plate and aspirate excess media and wash once with PBS to remove culture media. Place organoids in the middle of the well and using a scalpel, mince organoids thoroughly.</li>
	<li>Add enzyme mix 1 and place at 37 &#176;C, 20% O2 and 5% CO2, shaking at 90 rpm for 10-15 min.</li>
	<li>Using a 1000 &#181;L pipette tip, resuspend organoids by pipetting up to 10x. Add enzyme mix 2 and mix it well by pipetting using a 1000 &#181;L pipette tip. Place at 37 &#176;C, 20% O2 and 5% CO2, shaking at 90 rpm for 10-15 min.</li>
	<li>Stop the dissociation process by resuspending the cell solution in 10 mL of cold STOP solution. Filter cell solution using a 70 &#181;M cell strainer. Centrifuge the filtered cell solution for 10 min at 300 x g at 4 &#176;C to form a pellet.</li>
	<li>Aspirate media, leaving a cell pellet and resuspend cells in 4 mL of cold 0.04% BSA. Filter cell solution using a 40 &#181;M cell strainer and count cells using a cell counter.</li>
	<li>Centrifuge the cell solution for 10 min at 300 x g at 4 &#176;C. Aspirate BSA solution, leaving a cell pellet. Resuspend cells according to a microfluidics-based-snRNA-seq kit manual in NSB+ medium.</li>
</ol>
3. Isolation of single nuclei from organoids
<ol>
	<li>Transfer organoids into chilled PBS and cut each organoid into 4 pieces. Wash organoids 2x with chilled PBS.</li>
	<li>Cut with scissors the pipette tip of a 1000 &#181;L pipette and transfer organoid pieces into a 2 mL douncer. Aspirate PBS completely and add 1 mL of NP40 lysis buffer.</li>
	<li>Dounce 3x with dounce pestle A and 3x with dounce pestle B. Transfer suspension into a 15 mL centrifuge tube. Wash douncer with an additional 1 mL of NP40 lysis buffer and transfer into a 15 mL centrifuge tube. Incubate for 5 min at RT and subsequently centrifuge suspension for 5 min at 500 x g.</li>
	<li>Aspirate supernatant leaving 50 &#181;L of supernatant behind. Add 1 mL of NSB+ without disturbing the pellet. Resuspend after 5 min of incubation.</li>
	<li>Layer suspension on top of a Percoll gradient (bottom layer: 1 mL of NSB + and 250 &#181;L of Percoll, middle layer: 1 mL of NSB+ and 188 &#181;L of Percoll, top layer: 1 mL of NSB+ and 125 &#181;L of Percoll). (Critical) Perform layering very carefully to avoid layer mixing.</li>
	<li>Centrifuge for 5 min at 500 x g at 4 &#176;C, aspirate supernatant and resuspend in NSB+. Filter nuclei solution using a 40 &#181;M cell strainer.</li>
	<li>Stain and count nuclei using DAPI and a Neubauer Chamber. Resuspend nuclei according to a microfluidics-based scRNA-seq kit manual.</li>
</ol>
4. Library preparation and sequencing
<ol>
	<li>Load 10,000 cells and nuclei into a microfluidics system and generate the library according to the manual of microfluidics based scRNA-seq kit (Table of Materials).</li>
	<li>Sequence the libraries with an estimated 6 x 108 reads per snRNA-seq library and 1.6 x 108 reads per scRNA-seq library, resulting in a sequencing saturation of 87% for the snRNA-seq dataset and 36% sequencing saturation for the scRNA-seq dataset.</li>
</ol>
5. Analysis
<ol>
	<li>Carry out analysis of sequencing using a data analysis pipeline for scRNA-seq and snRNA-seq and align against the human GRCh38 genome. Subject the count matrix generated by this pipeline to further analysis using the R-package Seurat (Version 4.1.1)25.</li>
	<li>Create the Seurat object by considering genes that were represented in at least 5 cells and incorporating cells that contained at least 300 genes. Perform additional quality control filtering by retaining cells that contained more than 1000 genes but less than 4000 genes for the scRNA-seq dataset and more than 800, but less than 4000 genes per nuclei for the snRNA-seq dataset. Exclude datasets cells and nuclei exceeding 5% mitochondrial reads.</li>
	<li>To mitigate batch effects between both methods, integrate both filtered datasets, normalize and visualize via Uniform Manifold Approximation and Projection (UMAP). Omit cluster 1 which displays low unique molecular identifier (UMI) and gene counts. Afterwards, for the clusters assign cell identities based on known marker genes and the 30-day-old organoid dataset from Ana Uzquiano et. al. (Supplementary Coding File 1)18.</li>
</ol>

Single Cell and Nuclei Extraction from Human Brain Organoids

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The authors declare no competing financial interests.

Transcriptomic analysis of single cells and single nuclei has emerged as a pivotal tool for understanding gene regulatory mechanisms within complex tissues. Both methods enable transcriptome studies of brain organoids. To ensure an overall successful experiment, the quality of the starting material is of high relevance. Therefore, it is necessary to cut the organoids regularly to prevent the formation of a necrotic core26. It is also possible to eliminate this issue with an Air-Liquid Interface cu...

Over the last years, organoid technologies have emerged as a promising tool to culture organ-like tissues1,2,3. Especially for organs that cannot be easily accessed, such as the human brain, organoids offer the opportunity to gain insights into&#160;development and disease manifestation4. As such, brain organoids have been widely used as an experimental model to investigate various human brain disorders, including developmental, psychiatric, or even neurodegenerative diseases4,5,6.
With the advent of single-cell transcriptome profiling technologies, primary human tissues and complex in vitro models could be studied with an unprecedented level of granularity, providing mechanistic insights into gene expression changes on the level of cell subpopulations in health and disease and informing about new putative therapeutic targets7,8,9. The organoid field has progressed by utilizing single-cell transcriptome profiling to assess cellular composition, reproducibility and the fidelity of brain organoid technologies10,11,12. Single-cell RNA-sequencing (scRNA-seq) enabled cell classification and the identification of genetic dysregulation in diseased organoids13,14. Importantly, it is the complexity of organoid tissues that necessitates the implementation of techniques that enable the profiling of individual cells. Characterization of organoids using methods such as bulk transcriptome profiling (bulk RNA sequencing) leads to masked cellular heterogeneity and gene expression profiles which are averaged across all types of cells within the complex tissue, ultimately limiting our understanding of ongoing processes during organoid development in health and disease15,16,17. As scRNA-seq methods continue to advance, an increasing number of atlases are being created, exemplified by resources like the Allen Brain Atlas or the Single cell atlas of human brain organoids by Uzquiano et al.18.
Accomplishing successful scRNA-seq from brain organoids relies on effective isolation and capture of intact cells. As the dissociation of brain organoids to obtain individual cells is based on enzymatic digestion, it can influence gene expression patterns by inducing stress and cell damage19,20. Hence, the dissociation of the tissue into individual cells is the most crucial step. An alternative approach is single-nucleus RNA sequencing (snRNA-seq), which facilitates the enzyme-free extraction of nuclei from both, fresh and frozen, tissue21,22. However, the isolation of nuclei from a tissue poses other challenges such as the enrichment of cell types of interest and the low RNA content of nuclei in comparison to cells.
Transcriptome studies of brain organoids are commonly conducted using scRNA-seq10,18,23. However, the isolation of single nuclei might provide an orthogonal and supplemental method to investigate the transcriptomic profile of organoids. Here, we introduce a toolbox for scRNA- and snRNA-seq for brain organoids and discuss the critical points for obtaining the best quality sequencing data.

<table><tbody><tr><td>1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H&lt;sub&gt;2&lt;/sub&gt;O (DTT)</td><td>Sigma</td><td>&amp;nbsp;43816-10ML</td><td></td></tr><tr><td>1.5 ml DNA low binding tubes&amp;nbsp;</td><td>VWR</td><td>525-0130</td><td>microcentrifuge tube</td></tr><tr><td>10x Cellranger pipeline&amp;nbsp;</td><td></td><td></td><td>analysis pipline</td></tr><tr><td>15 ml Falcon</td><td>Falcon</td><td></td><td>Centrifuge tube</td></tr><tr><td>2-Mercaptoethanol (BME)</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>50 ml Falcon</td><td>Falcon</td><td></td><td>Centrifuge tube</td></tr><tr><td>A83-01</td><td>Bio Technologies</td><td>379762</td><td></td></tr><tr><td>Antibiotic/Antimycotic Solution (100X)</td><td>Life Technologies</td><td>15240062</td><td></td></tr><tr><td>B-27 Plus Supplement</td><td>Life Technologies</td><td>17504044</td><td></td></tr><tr><td>B-27 Supplement without vitamin A</td><td>Life Technologies</td><td>12587010</td><td></td></tr><tr><td>Bovine serum albumin, fatty acid free (BSA)</td><td>Sigma Aldrich</td><td>A8806-5G&amp;nbsp;</td><td></td></tr><tr><td>cAMP</td><td>Biogems</td><td>6099240</td><td></td></tr><tr><td>cAMP</td><td>Biogems</td><td>6099240</td><td></td></tr><tr><td>C-CHIP NEUBAUER IMPROVED</td><td>VWR</td><td>DHC-N01</td><td></td></tr><tr><td>Cell strainer 40 &amp;micro;m</td><td>Neolab</td><td>352340</td><td></td></tr><tr><td>Cell strainer 70 &amp;micro;m (white) Nylon</td><td>Sigma</td><td>CLS431751-50EA</td><td></td></tr><tr><td>Chromium Controller &amp;amp; Next GEM Accessory Kit</td><td>10X Genomics</td><td>1000204</td><td></td></tr><tr><td>Chromium Next GEM Chip G Single Cell Kit, 16 rxns</td><td>10X Genomics</td><td>1000127</td><td></td></tr><tr><td>Chromium Next GEM Single Cell 3&#039; Kit v3.1</td><td>10X Genomics</td><td>1000268</td><td></td></tr><tr><td>Complete,&amp;nbsp; EDTA-free Protease Inhibitor Cocktaill</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>DAPI</td><td>MERCK Chemicals</td><td>0000001722</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320074</td><td></td></tr><tr><td>Dounce tissue grinder set 2 mL complete</td><td>Sigma Aldrich</td><td>10536355</td><td></td></tr><tr><td>Essential E8 Flex Medium</td><td>Life Technologies</td><td>A2858501</td><td></td></tr><tr><td>EVE Cell Counting Slides</td><td>VWR</td><td>EVS-050 ( 734-2676)</td><td></td></tr><tr><td>Foetal bovine serum tetracycline free (FBS)</td><td>PAN Biotech</td><td>P30-3602</td><td></td></tr><tr><td>Geltrex LDEV-Free (coating)</td><td>Life Technologies</td><td>A1413302&amp;nbsp;</td><td></td></tr><tr><td>gentleMACS</td><td>Miltenyi Biotec</td><td></td><td>dissociation maschine</td></tr><tr><td>GlutaMAX supplements</td><td>Life Technologies</td><td>35050038</td><td></td></tr><tr><td>Heparin sodium cell culture tested</td><td>Sigma</td><td>H3149-10KU</td><td></td></tr><tr><td>human recombinant BDNF</td><td>StemCell Technologies</td><td>78005.3</td><td></td></tr><tr><td>human recombinant GDNF</td><td>StemCell Technologies</td><td>78058.3</td><td></td></tr><tr><td>Insulin Solution Human</td><td>Sigma Aldrich</td><td>I2643-25MG</td><td></td></tr><tr><td>Knockout serum replacement</td><td>Life Technologies</td><td>10828028</td><td></td></tr><tr><td>LDN193189 Hydrochloride 98%</td><td>Sigma Aldrich</td><td>130-106-540</td><td></td></tr><tr><td>MEM non-essential amino acid (100x)</td><td>Sigma Aldrich</td><td>M7145-100ml</td><td></td></tr><tr><td>MgCl2 Magnesium Chloride (1M) RNAse free</td><td>Thermo Scientific</td><td>AM9530G</td><td></td></tr><tr><td>mTeSR Plus</td><td>StemCell Technologies</td><td>100-0276</td><td>stem cell medium</td></tr><tr><td>mTeSR1</td><td>StemCell Technologies</td><td>85850</td><td>stem cell medium</td></tr><tr><td>N2 Supplement&amp;nbsp;</td><td>StemCell Technologies</td><td>17502048</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit</td><td>Miltenyi Biotec B.V. &amp;amp; Co. KG</td><td>130-092-628</td><td></td></tr><tr><td>Neurobasal Plus</td><td>Life Technologies</td><td>A3582901</td><td></td></tr><tr><td>NextSeq500 system</td><td>Illumina</td><td></td><td>Sequencer</td></tr><tr><td>NP-40 Surfact-Amps Detergent Solution</td><td>Life Technologies</td><td>28324</td><td></td></tr><tr><td>PBS Dulbecco&amp;rsquo;s</td><td>Invitrogen</td><td>14190169</td><td></td></tr><tr><td>PenStrep (Penicillin - Streptomycin)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Percoll</td><td>Th. Geyer</td><td>10668276</td><td></td></tr><tr><td>Pluronic (R) F-127</td><td>Sigma Aldrich</td><td>P2443-1KG</td><td></td></tr><tr><td>RiboLock RNase Inhibitor</td><td>Life Technologies&amp;nbsp;</td><td>EO0382</td><td></td></tr><tr><td>Rock Inhibitor (Y-27632 dihydrochloride) SB</td><td>Biomol</td><td>Cay10005583-10</td><td></td></tr><tr><td>SB 431542&amp;nbsp;</td><td>Biogems</td><td>3014193</td><td></td></tr><tr><td>Sodium chloride NaCl (5M), RNase-free-100 mL</td><td>Invitrogen</td><td>AM9760G</td><td></td></tr><tr><td>StemFlex Medium</td><td>Thermo Scientific</td><td>A3349401</td><td>stem cell medium</td></tr><tr><td>StemMACS iPS-Brew XF</td><td>Miltenyi Biotec</td><td>130-104-368</td><td>stem cell medium</td></tr><tr><td>TC-Platte 96 Well, round bottom</td><td>Sarstedt</td><td>83.3925.500</td><td></td></tr><tr><td>TISSUi006-A</td><td>TissUse GmbH</td><td>https://hpscreg.eu/cell-line/TISSUi006-A</td><td></td></tr><tr><td>Trypan Blue</td><td>T8154-20ml</td><td>Sigma</td><td></td></tr><tr><td>TrypLE Express Enzyme, no phenol red</td><td>Life Technologies</td><td>12604013</td><td>Trypsin-based reagent</td></tr><tr><td>UltraPure 1M Tris-HCl Buffer, pH 7.5</td><td>Life Technologies</td><td>15567027</td><td></td></tr><tr><td>XAV939</td><td>Enzo Life sciences</td><td>BML-WN100-0005</td><td></td></tr></tbody></table>

generation and downstream analysis of single-cell and single-nuclei transcriptomes in brain organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

To investigate cell type composition of brain organoids using scRNA-seq and snRNA-seq, brain organoids were harvested after 30 days of culture as organoids at this stage already exhibit neuroepithelial loops consisting of progenitors surrounded by intermediate progenitors and early stage neurons4,18. Monitoring the quality of the organoids throughout growth and culturing is essential for obtaining reliable single-cell and single-nucleus data.
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Watch this Scientific Journal Video about Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids at JoVE.com

Author Spotlight: Integrating Organoid Models with Single-Cell and Spatial Transcriptomics Technologies

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

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Research

JoVE Journal

Developmental Biology

2.1K Views.  Max Delbrück Center for Molecular Medicine (MDC). Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Video: Author Spotlight: Integrating Organoid Models with Single-Cell and Spatial Transcriptomics Technologies

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 ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

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 ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

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 Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Biology

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent ...

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

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

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

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

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research