Development and Characterization of Human Cerebral Organoids: An Optimized Protocol
Open accessAbstract
Studies of human neurodevelopmental disorders and stem cell–based regenerative transplants have been hampered by the lack of a model of the developing human brain. Stem cell–derived neurons suffer major limitations, including the ability to recapitulate the 3-dimensional architecture of a brain tissue and the representation of multiple layers and cell types that contribute to the overall brain functions in vivo. Recently, cerebral organoid technology was introduced; however, such technology is still in its infancy, and its low reproducibility and limitations significantly reduce the reliability of such a model as it currently exists, especially considering the complexity of cerebral-organoid protocols. Here we have tested and compared multiple protocols and conditions for growth of organoids, and we describe an optimized methodology, and define the necessary and sufficient factors that support the development of optimal organoids. Our optimization criteria included organoids’ overall growth and size, stratification and representation of the various cell types, inter-batch variability, analysis of neuronal maturation, and even the cost of the procedure. Importantly, this protocol encompasses a plethora of technical tips that allow researchers to easily reproduce it and obtain reliable organoids with the least variability, and showcases a robust array of approaches to characterize successful organoids. This optimized protocol provides a reliable system for genetic or pharmacological (drug development) screens and may enhance understanding and therapy of human neurodevelopmental disorders, including harnessing the therapeutic potential of stem cell–derived transplants.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The success rate of on-chip tubular organoid formation is around 60%, comparable with the intrinsic success rate (70%) of the cerebral organoid protocol.
“Overall, we observe a success rate of on-chip tubular organoid formation around 60%, a rate comparable with the intrinsic success rate (70%) with the cerebral organoid protocol.52”
From Tubular human brain organoids to model microglia-mediated neuroinflammation (2021) · cites this paper as [52]
The Procedures section describes measuring successful embryoid-body formation, and the Characterization section describes successful cerebral organoids, but neither passage gives a 70% success rate. Searches for “70% success rate” and cerebral-organoid formation efficiency found no passage establishing that figure. The on-chip 60% rate is presented as the citing authors’ own observation, not a result attributed to this source. (LLM, conf=0.72)
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling
Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009) · Nature Biotechnology · doi:10.1038/nbt.1529 · Cited by 3,620 · see citing works
Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure
Qian, X., Nguyen, H. N., Song, M. M., Hadiono, C., Ogden, S. C., Hammack, C., Yao, B., Hamersky, G. R., Jacob, F., Zhong, C., et al. (2016) · Cell · doi:10.1016/j.cell.2016.04.032 · Cited by 2,127 · see citing works
Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture
Paşca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J.-Y., O'Rourke, N. A., Nguyen, K. D., et al. (2015) · Nature Methods · doi:10.1038/nmeth.3415 · Cited by 1,609 · see citing works
Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S., Matsumura, M., Wataya, T., Nishiyama, A., Muguruma, K., & Sasai, Y. (2008) · Cell Stem Cell · doi:10.1016/j.stem.2008.09.002 · Cited by 1,522 · see citing works
Neurogenic radial glia in the outer subventricular zone of human neocortex
Hansen, D. V., Lui, J. H., Parker, P. R. L., & Kriegstein, A. R. (2010) · Nature · doi:10.1038/nature08845 · Cited by 1,388 · see citing works
Modeling autism-relevant behavioral phenotypes in rats and mice: Do ‘autistic’ rodents exist?
Servadio, M., Vanderschuren, L. J. M. J., & Trezza, V. (2015) · Behavioural Pharmacology · doi:10.1097/fbp.0000000000000163 · Cited by 76 · see citing works
Flagged first, then by citations
Research works printed but not linked: 19
- [1]Kazdoba TM Leach PT Yang M Silverman JL Solomon M Crawley JN Translational mouse models of autism: advancing toward pharmacological therapeutics. Curr Top Behav Neurosci. 2016; 28: 1– 52. 27305922 PMC5116923
- [3]Nestor MW Phillips AW Artimovich E Nestor JE Hussman JP Blatt GJ Human inducible pluripotent stem cells and autism spectrum disorder: emerging technologies. Autism Res. 2016; 9(5): 513– 535. 26426199
- [5]Watanabe K Kamiya D Nishiyama A Katayama T Nozaki S Kawasaki H Watanabe Y Mizuseki K Sasai Y Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci. 2005; 8(3): 288– 296. 15696161
- [6]Eiraku M Sasai Y Self-formation of layered neural structures in three-dimensional culture of ES Cells. Curr Opin Neurobiol. 2012; 22(5): 768– 777. 22405989
- [7]Shi Y Kirwan P Smith J Robinson HP Livesey FJ Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci. 2012; 15(3): 477– 486. 22306606 PMC3882590
- [8]Mariani J Simonini MV Palejev D Tomasini L Coppola G Szekely AM Horvath TL Vaccarino FM Modeling human cortical development in vitro using induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2012; 109(31): 12770– 12775. 22761314 PMC3411972
- [9]Sasai Y Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell. 2013; 12(5): 520– 530. 23642363
- [11]Mariani J Coppola G Zhang P Abyzov A Provini L Tomasini L Amenduni M Szekely A Palejev D Wilson M Gerstein M Grigorenko EL Chawarska K Pelphrey KA Howe JR Vaccarino FM FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell. 2015; 162(2): 375– 390. 26186191 PMC4519016
- [12]Lancaster MA Renner M Martin CA Wenzel D Bicknell LS Hurles ME Homfray T Penninger JM Jackson AP Knoblich JA Cerebral organoids model human brain development and microcephaly. Nature. 2013; 501(7467): 373– 379. 23995685 PMC3817409
- [14]Garcez PP Loiola EC Madeiro da Costa R Higa LM Trindade P Delvecchio R Nascimento JM Brindeiro R Tanuri A Rehen SK Zika virus impairs growth in human neurospheres and brain organoids. Science. 2016; 352(6287): 816– 818. 27064148
- [15]Dang J Tiwari SK Lichinchi G Qin Y Patil VS Eroshkin AM Rana TM Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell. 2016; 19(2): 258– 265. 27162029 PMC5116380
- [16]Gabriel E Ramani A Karow U Gottardo M Natarajan K Gooi LM Goranci-Buzhala G Krut O Peters F Nikolic M Kuivanen S Korhonen E Smura T Vapalahti O Papantonis A Schmidt-Chanasit J Riparbelli M Callaini G Krönke M Utermöhlen O Gopalakrishnan J Recent Zika virus isolates induce premature differentiation of neural progenitors in human brain organoids. Cell Stem Cell. 2017; 20(3): 397– 406. 28132835
- [17]Lancaster MA Knoblich JA Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014; 9(10): 2329– 2340. 25188634 PMC4160653
- [18]Watanabe K Ueno M Kamiya D Nishiyama A Matsumura M Wataya T Takahashi JB Nishikawa S Nishikawa S Muguruma K Sasai Y A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007; 25(6): 681– 686. 17529971
- [20]Gaspard N Bouschet T Hourez R Dimidschstein J Naeije G van den Ameele J Espuny-Camacho I Herpoel A Passante L Schiffmann SN Gaillard A Vanderhaeghen P An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature. 2008; 455(7211): 351– 357. 18716623
- [22]Yi F Danko T Botelho SC Patzke C Pak C Wernig M Südhof TC Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons. Science. 2016; 352(6286): aaf2669. PMC4901875 26966193
- [23]Menendez L Yatskievych TA Antin PB Dalton S Wnt signaling and a SMAD pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc Natl Acad Sci U S A. 2011; 108(48): 19240– 19245. 22084120 PMC3228464
- [24]Kent L. Culture and maintenance of human embryonic stem cells. J Vis Exp. 2009; 34, pii:1427. PMC3152239 20029367
- [25]Kitsberg D Human embryonic stem cells for tissue engineering. Methods Mol Med. 2007; 140: 33– 65. 18085202
Cited by
Sorted by citations
Tubular human brain organoids to model microglia-mediated neuroinflammation
Ao, Z., Cai, H., Wu, Z., Song, S., Karahan, H., Kim, B., Lu, H.-C., Kim, J., Mackie, K., & Guo, F. (2021) · Lab on a Chip · doi:10.1039/d1lc00030f · Cited by 87 · see citing works
- Authors
- Yakoub, A. M., & Sadek, M.
- Affiliations
- Stanford Medicine (US)
- University of Illinois Chicago (US)
- Corresponding author
- Yakoub, Abraam M.
- ORCID
- Yakoub, Abraam M.
- Venue
- Cell Transplantation
- Publisher
- SAGE Publications
- Published
- 2018-03
- Volume
- 27
- Issue
- 3
- Pages
- 393-406
- ISSN
- 0963-6897 (ISSN-L)
- 1555-3892
- PMC
- PMC6038047
- OpenAlex
- W2800784717
- Cited by
- 47
- References
- 6
- Citations by year
- 2026: 1 · 2025: 7 · 2024: 3 · 2023: 9 · 2022: 5 · 2021: 6 · 2020: 7 · 2019: 9
- FWCI
- 1.96
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Transplantation (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Keywords
- brain organoids, human brain development, neurodevelopmental disorders, reproducibility, neuronal maturation, batch-to-batch variation, stem cell-derived neurons, drug screening model, organoid characterization
- MeSH terms
- Brain, Humans, Organoids, Stem Cells, Neurodevelopmental Disorders
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
