Abstract
The advent of human-induced pluripotent stem cell (iPSC)-derived three-dimensional (3D) cerebral organoids provides unprecedented opportunities of modeling human brains in states of health and disorder. Emerging data supports that cerebral organoids allow for more relevant in vitro systems for studying the human brain system and diseases than the current widely used 2D monolayer cell culture. Thus, the ability to isolate, culture, and maintain human brain cells from cerebral organoids is highly needed, particularly for studies on organoid-derived cell-type-specific signaling and their electrophysiological properties. Here we present a protocol to isolate and culture brain cells from 2-month human iPSC-derived cerebral organoids. The dissociation and plating of cells from organoids takes 3–4 h. The dissociated cells can be maintained in culture for up to at least 3 weeks. Some cells expressed the neuron-specific marker microtubule-associated protein 2 and exhibited spontaneous action potentials.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Akhtar A (2015) The flaws and human harms of animal experimentation. Camb Q Healthc Ethics 24(4):407–419. https://doi.org/10.1017/S0963180115000079
Traynor BJ, Bruijn L, Conwit R, Beal F, O'Neill G, Fagan SC, Cudkowicz ME (2006) Neuroprotective agents for clinical trials in ALS: a systematic assessment. Neurology 67(1):20–27. https://doi.org/10.1212/01.wnl.0000223353.34006.54
Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, Finnerty CC, Lopez CM, Honari S, Moore EE, Minei JP, Cuschieri J, Bankey PE, Johnson JL, Sperry J, Nathens AB, Billiar TR, West MA, Jeschke MG, Klein MB, Gamelli RL, Gibran NS, Brownstein BH, Miller-Graziano C, Calvano SE, Mason PH, Cobb JP, Rahme LG, Lowry SF, Maier RV, Moldawer LL, Herndon DN, Davis RW, Xiao W, Tompkins RG (2013) Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A 110(9):3507–3512. https://doi.org/10.1073/pnas.1222878110
Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379. https://doi.org/10.1038/nature12517
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872. https://doi.org/10.1016/j.cell.2007.11.019
Logan S, Arzua T, Canfield SG, Seminary ER, Sison SL, Ebert AD, Bai X (2019) Studying human neurological disorders using induced pluripotent stem cells: from 2D monolayer to 3D organoid and blood brain barrier models. Compr Physiol 9(2):565–611. https://doi.org/10.1002/cphy.c180025
Horikoshi Y, Yan Y, Terashvili M, Wells C, Horikoshi H, Fujita S, Bosnjak ZJ, Bai X (2019) Fatty acid-treated induced pluripotent stem cell-derived human cardiomyocytes exhibit adult cardiomyocyte-like energy metabolism phenotypes. Cell 8(9). https://doi.org/10.3390/cells8091095
Kikuchi C, Bienengraeber M, Canfield S, Koopmeiner A, Schafer R, Bosnjak ZJ, Bai X (2015) Comparison of cardiomyocyte differentiation potential between type 1 diabetic donor- and nondiabetic donor-derived induced pluripotent stem cells. Cell Transplant 24(12):2491–2504. https://doi.org/10.3727/096368914X685762
Logan S, Arzua T, Yan Y, Jiang C, Liu X, Yu LK, Liu QS, Bai X (2020) Dynamic characterization of structural, molecular, and electrophysiological phenotypes of human-induced pluripotent stem cell-derived cerebral organoids, and comparison with Fetal and adult gene profiles. Cell 9(5). https://doi.org/10.3390/cells9051301
Ormel PR, Vieira de Sa R, van Bodegraven EJ, Karst H, Harschnitz O, Sneeboer MAM, Johansen LE, van Dijk RE, Scheefhals N, Berdenis van Berlekom A, Ribes Martinez E, Kling S, MacGillavry HD, van den Berg LH, Kahn RS, Hol EM, de Witte LD, Pasterkamp RJ (2018) Microglia innately develop within cerebral organoids. Nat Commun 9(1):4167. https://doi.org/10.1038/s41467-018-06684-2
Yakoub AM (2019) Cerebral organoids exhibit mature neurons and astrocytes and recapitulate electrophysiological activity of the human brain. Neural Regen Res 14(5):757–761. https://doi.org/10.4103/1673-5374.249283
Karzbrun E, Reiner O (2019) Brain organoids-A bottom-up approach for studying human neurodevelopment. Bioengineering (Basel) 6(1). https://doi.org/10.3390/bioengineering6010009
Dezonne RS, Sartore RC, Nascimento JM, Saia-Cereda VM, Romao LF, Alves-Leon SV, de Souza JM, Martins-de-Souza D, Rehen SK, Gomes FC (2017) Derivation of functional human astrocytes from cerebral organoids. Sci Rep 7:45091. https://doi.org/10.1038/srep45091
Logan S, Arzua T, Yan Y, Jiang C, Liu X, Yan L, Liu Q, Bai X (2020) Comparison of genome-wide expression profiles among human induced pluripotent stem cell-derived cerebral organoids, fetal and adult brains. Cells in review
Arzua T, Yan Y, Jiang C, Logan S, Wells C, Kumar S, Bai X (2020) Modeling alcohol-induced neurotoxicity using human induced pluripotent stem cell-derived three-dimensional cerebral organoids. International Society for Stem Cell Research (ISSCR) 2020 annual meeting
Wang H (2018) Modeling neurological diseases with human brain organoids. Front Synaptic Neurosci 10:15. https://doi.org/10.3389/fnsyn.2018.00015
Pasca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD, Huber N, Kim CH, Park JY, O'Rourke NA, Nguyen KD, Smith SJ, Huguenard JR, Geschwind DH, Barres BA, Pasca SP (2015) Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods 12(7):671–678. https://doi.org/10.1038/nmeth.3415
Nowakowski TJ, Pollen AA, Di Lullo E, Sandoval-Espinosa C, Bershteyn M, Kriegstein AR (2016) Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem cells. Cell Stem Cell 18(5):591–596. https://doi.org/10.1016/j.stem.2016.03.012
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, Kronke M, Utermohlen O, Gopalakrishnan J (2017) Recent Zika virus isolates induce premature differentiation of neural progenitors in human brain organoids. Cell Stem Cell 20(3):397–406 e395. https://doi.org/10.1016/j.stem.2016.12.005
Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, Hamersky GR, Jacob F, Zhong C, Yoon KJ, Jeang W, Lin L, Li Y, Thakor J, Berg DA, Zhang C, Kang E, Chickering M, Nauen D, Ho CY, Wen Z, Christian KM, Shi PY, Maher BJ, Wu H, Jin P, Tang H, Song H, Ming GL (2016) Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165(5):1238–1254. https://doi.org/10.1016/j.cell.2016.04.032
Li Y, Muffat J, Omer A, Bosch I, Lancaster MA, Sur M, Gehrke L, Knoblich JA, Jaenisch R (2017) Induction of expansion and folding in human cerebral organoids. Cell Stem Cell 20(3):385–396 e383. https://doi.org/10.1016/j.stem.2016.11.017
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 (2015) FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162(2):375–390. https://doi.org/10.1016/j.cell.2015.06.034
Lee CT, Bendriem RM, Wu WW, Shen RF (2017) 3D brain organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders. J Biomed Sci 24(1):59. https://doi.org/10.1186/s12929-017-0362-8
Li R, Sun L, Fang A, Li P, Wu Q, Wang X (2017) Recapitulating cortical development with organoid culture in vitro and modeling abnormal spindle-like (ASPM related primary) microcephaly disease. Protein Cell 8(11):823–833. https://doi.org/10.1007/s13238-017-0479-2
Trujillo CA, Muotri AR (2018) Brain organoids and the study of neurodevelopment. Trends Mol Med 24(12):982–990. https://doi.org/10.1016/j.molmed.2018.09.005
Quadrato G, Nguyen T, Macosko EZ, Sherwood JL, Min Yang S, Berger DR, Maria N, Scholvin J, Goldman M, Kinney JP, Boyden ES, Lichtman JW, Williams ZM, McCarroll SA, Arlotta P (2017) Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545(7652):48–53. https://doi.org/10.1038/nature22047
Cugola FR, Fernandes IR, Russo FB, Freitas BC, Dias JL, Guimaraes KP, Benazzato C, Almeida N, Pignatari GC, Romero S, Polonio CM, Cunha I, Freitas CL, Brandao WN, Rossato C, Andrade DG, Faria Dde P, Garcez AT, Buchpigel CA, Braconi CT, Mendes E, Sall AA, Zanotto PM, Peron JP, Muotri AR, Beltrao-Braga PC (2016) The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534(7606):267–271. https://doi.org/10.1038/nature18296
Zhang B, Chu H, Han S, Shuai H, Deng J, Hu Y, Gong H, Lee A, Zou Z, Yau T, Wu W, Hung H, Chan J, Yuen K, Huang J (2020) SARS-CoV-2 infects human neural progenitor cells and brain organoids. Cell Rep 0:1–4. https://doi.org/10.1038/s41422-020-0390-x
Acknowledgments
This work was supported by the National Institute of Health R01 GM112696 (to X. Bai).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media New York
About this protocol
Cite this protocol
Yan, Y., Arzua, T., Logan, S., Bai, X. (2020). Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells. In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2020_328
Download citation
DOI: https://doi.org/10.1007/7651_2020_328
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2118-9
Online ISBN: 978-1-0716-2119-6
eBook Packages: Springer Protocols