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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

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 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.

Introduction

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 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,

Protocol

The described protocol is performed in a biosafety level 1 laboratory of the Max Delbrück 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 differen.......

Representative Results

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.

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H2O (DTT)Sigma 43816-10ML
1.5 ml DNA low binding tubes VWR525-0130microcentrifuge tube
10x Cellranger pipeline analysis pipline
15 ml FalconFalconCentrifuge tube
2-Mercaptoethanol (BME)Life Technologies21985023
50 ml FalconFalconCentrifuge tube
A83-01Bio Technologies379762
Antibiotic/Antimycotic Solution (100X)Life Technologies15240062
B-27 Plus SupplementLife Technologies17504044
B-27 Supplement without vitamin ALife Technologies12587010
Bovine serum albumin, fatty acid free (BSA)Sigma AldrichA8806-5G 
cAMPBiogems6099240
cAMPBiogems6099240
C-CHIP NEUBAUER IMPROVEDVWRDHC-N01
Cell strainer 40 µmNeolab352340
Cell strainer 70 µm (white) NylonSigmaCLS431751-50EA
Chromium Controller & Next GEM Accessory Kit10X Genomics1000204
Chromium Next GEM Chip G Single Cell Kit, 16 rxns10X Genomics1000127
Chromium Next GEM Single Cell 3' Kit v3.110X Genomics1000268
Complete,  EDTA-free Protease Inhibitor CocktaillRoche11873580001
DAPIMERCK Chemicals0000001722
DMEM/F12Life Technologies11320074
Dounce tissue grinder set 2 mL completeSigma Aldrich10536355
Essential E8 Flex MediumLife TechnologiesA2858501
EVE Cell Counting SlidesVWREVS-050 ( 734-2676)
Foetal bovine serum tetracycline free (FBS)PAN BiotechP30-3602
Geltrex LDEV-Free (coating)Life TechnologiesA1413302 
gentleMACSMiltenyi Biotecdissociation maschine
GlutaMAX supplementsLife Technologies35050038
Heparin sodium cell culture testedSigmaH3149-10KU
human recombinant BDNFStemCell Technologies78005.3
human recombinant GDNFStemCell Technologies78058.3
Insulin Solution HumanSigma AldrichI2643-25MG
Knockout serum replacementLife Technologies10828028
LDN193189 Hydrochloride 98%Sigma Aldrich130-106-540
MEM non-essential amino acid (100x)Sigma AldrichM7145-100ml
MgCl2 Magnesium Chloride (1M) RNAse freeThermo ScientificAM9530G
mTeSR PlusStemCell Technologies100-0276stem cell medium
mTeSR1StemCell Technologies85850stem cell medium
N2 Supplement StemCell Technologies17502048
Neural Tissue Dissociation KitMiltenyi Biotec B.V. & Co. KG130-092-628
Neurobasal PlusLife TechnologiesA3582901
NextSeq500 systemIlluminaSequencer
NP-40 Surfact-Amps Detergent SolutionLife Technologies28324
PBS Dulbecco’sInvitrogen14190169
PenStrep (Penicillin - Streptomycin)Life Technologies15140122
PercollTh. Geyer10668276
Pluronic (R) F-127Sigma AldrichP2443-1KG
RiboLock RNase InhibitorLife Technologies EO0382
Rock Inhibitor (Y-27632 dihydrochloride) SBBiomolCay10005583-10
SB 431542 Biogems3014193
Sodium chloride NaCl (5M), RNase-free-100 mLInvitrogenAM9760G
StemFlex MediumThermo ScientificA3349401stem cell medium
StemMACS iPS-Brew XFMiltenyi Biotec130-104-368stem cell medium
TC-Platte 96 Well, round bottomSarstedt83.3925.500
TISSUi006-ATissUse GmbHhttps://hpscreg.eu/cell-line/TISSUi006-A
Trypan BlueT8154-20mlSigma
TrypLE Express Enzyme, no phenol redLife Technologies12604013Trypsin-based reagent
UltraPure 1M Tris-HCl Buffer, pH 7.5Life Technologies15567027
XAV939Enzo Life sciencesBML-WN100-0005

References

  1. Finkbeiner, S. R., et al. Stem cell-derived human intestinal organoids as an infection model for Rotaviruses. mBio. 3 (4), e00159-e00212 (2012).
  2. Freedman, B. S., et al.

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Single cell TranscriptomicsSingle nucleus TranscriptomicsBrain OrganoidsCell Type IdentificationGene ExpressionCell LineageDevelopmental ProcessesMolecular Mechanisms

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