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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H2O (DTT)</td>
			<td>Sigma</td>
			<td>&#160;43816-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml DNA low binding tubes&#160;</td>
			<td>VWR</td>
			<td>525-0130</td>
			<td>microcentrifuge tube</td>
		</tr>
		<tr>
			<td>10x Cellranger pipeline&#160;</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&#160;</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 &#181;m</td>
			<td>Neolab</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#181;m (white) Nylon</td>
			<td>Sigma</td>
			<td>CLS431751-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Controller &#38; 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&#39; Kit v3.1</td>
			<td>10X Genomics</td>
			<td>1000268</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete,&#160; 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&#160;</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&#160;</td>
			<td>StemCell Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit</td>
			<td>Miltenyi Biotec B.V. &#38; 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&#8217;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&#160;</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&#160;</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 ...

generation-downstream-analysis-single-cell-single-nuclei

Research

JoVE Journal

Developmental Biology

1.8K 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 ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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. ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.