Abstract
Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and large-scale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps (> 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a large number of neurons that are relevant for basic research, large-scale drug screening, and toxicological studies.
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Abbreviations
- BDNF:
-
Brain derived neurotrophic factor
- iPSCs:
-
Induced pluripotent stem cells
- MSN:
-
Medium spiny neuron
- NDM:
-
Neuronal differentiation medium
- NE:
-
Neuroectodermal
- pMSN:
-
Precursor of medium spiny neuron
References
Abdipranoto-Cowley A, Park JS, Croucher D, Danie J, Henshall S, Galbraith S, Mervin K, Vissel B (2009) Activin A is essential for neurogenesis following neurodegeneration. Stem Cells 27:1330–1346. https://doi.org/10.1002/stem.80
Alcalá-Barraza SR, Lee MS, Hanson LR, McDonald AA (2010) Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS. J Drug Target 18:179–190. https://doi.org/10.3109/10611860903318134
Arber C, Precious SV, Cambray S, Risner-Janiczek JR, Kelly C, Noakes Z, Fjodorova M, Heuer A, Ungless MA, Rodríguez TA, Rosser AE, Dunnett SB, Li M (2015) Activin A directs striatal projection neuron differentiation of human pluripotent stem cells. Development 142:1375–1386. https://doi.org/10.1242/dev.117093
Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL (2008) Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci USA. 105:16707–16712. https://doi.org/10.1073/pnas.0808488105
Baquet ZC, Gorski JA, Jones KR (2004) Early striatal dendrite deficits followed by neuron loss with advanced age in the absence of anterograde cortical brain-derived neurotrophic factor. J Neurosci 24:4250–4258. https://doi.org/10.1523/JNEUROSCI.3920-03.2004
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280. https://doi.org/10.1038/nbt.1529
Chuhma N, Tanaka KF, Hen R, Rayport S (2011) Functional connectome of the striatal medium spiny neuron. J Neurosci 31:1183–1192. https://doi.org/10.1523/JNEUROSCI.3833-10.2011
El-Akabawy G, Medina LM, Jeffries A, Price J, Modo M (2011) Purmorphamine increases DARPP-32 differentiation in human striatal neural stem cells through the hedgehog pathway. Stem Cells Dev 20:1873–1887. https://doi.org/10.1089/scd.2010.0282
Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L (2008) Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev 22:152–165. https://doi.org/10.1101/gad.1616208
Ericson J, Muhr J, Jessell TM, Edlund T (1995) Sonic hedgehog: a common signal for ventral patterning along the rostrocaudal axis of the neural tube. Int J Dev Biol 39:809–816
Gage FH, Coates PW, Palmer TD, Kuhn HG, Fisher LJ, Suhonen JO, Peterson DA, Suhr ST, Ray J (1995) Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA. 92:11879–11883. https://doi.org/10.1073/pnas.92.25.11879
Graybiel AM (2005) The basal ganglia: learning new tricks and loving it. Curr Opin Neurobiol 15:638–644. https://doi.org/10.1016/j.conb.2005.10.006
Grigor’eva EV, Shevchenko AI, Medvedev SP, Mazurok NA, Zhelezova AI, Zakian SM (2015) Induced pluripotent stem cells of Microtus levis x Microtus arvalis vole hybrids: conditions necessary for their generation and self-renewal. Acta Naturae 7:56–69
Grigor’eva EV, Malankhanova TB, Surumbayeva A, Minina JM, Morozov VV, Abramycheva NY, Illarioshkin SN, Malakhova AA, Zakian SM (2019) Generation of induced pluripotent stem cell line, ICGi007-A, by reprogramming peripheral blood mononuclear cells from a patient with Huntington’s disease. Stem Cell Res. https://doi.org/10.1016/j.scr.2018.101382
Haramoto M, Tatemoto H, Muto N (2008) Essential role of ascorbic acid in neural differentiation and development: high levels of ascorbic acid 2-glucoside effectively enhance nerve growth factor-induced neurite formation and elongation in PC12 Cells. J Health Sci 54:43–49. https://doi.org/10.1248/jhs.54.43
Hunt CPJ, Pouton CW, Haynes JM (2017) Characterising the developmental profile of hESC-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line. Neurochem Int 106:3–13. https://doi.org/10.1016/j.neuint.2017.01.003
Jeon I, Lee N, Li JY, Park IH, Park KS, Moon J, Shim SH, Choi C, Chang DJ, Kwon J, Oh SH, Shin DA, Kim HS, Do JT, Lee DR, Kim M, Kang KS, Daley GQ, Brundin P, Song J (2012) Neuronal properties, in vivo effects, and pathology of a Huntington’s disease patient-derived induced pluripotent stem cells. Stem Cells. 30:2054–2062. https://doi.org/10.1002/stem.1135
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28:31–40. https://doi.org/10.1016/s0896-6273(00)00083-0
Knowlton BJ, Mangels JA, Squire LR (1996) A neostriatal habit learning system in humans. Science 273:1399–1402. https://doi.org/10.1126/science.273.5280.1399
Lee H, Shamy GA, Elkabetz Y, Schofield CM, Harrsion NL, Panagiotakos G, Socci ND, Tabar V, Studer L (2007) Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells. 25:1931–1939. https://doi.org/10.1634/stemcells.2007-0097
Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang SC (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23:215–221. https://doi.org/10.1038/nbt1063
Liu H, Zhang S (2011) Specification of neuronal and glial subtypes from human pluripotent stem cells. Cell Mol Life Sci 68:3995–4008. https://doi.org/10.1007/s00018-011-0770-y
López-Toledano MA, Redondo C, Lobo MV, Reimers D, Herranz AS, Paíno CL, Bazán E (2004) Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C. J Histochem Cytochem 52:1177–1189. https://doi.org/10.1369/jhc.3A6244.2004
Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP (2008) Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev Biol 8:90. https://doi.org/10.1186/1471-213X-8-90
Ma L, Hu B, Liu Y, Vermilyea SC, Liu H, Gao L, Sun Y, Zhang X, Zhang SC (2012) Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice. Cell Stem Cell 10:455–464. https://doi.org/10.1016/j.stem.2012.01.021
Mattis VB, Svendsen SP, Ebert A, Svendsen CN et al (2012) Induced pluripotent stem cells from patients with Huntington’s disease show CAG-repeat-expansion-associated phenotypes. Cell Stem Cell 11:264–278. https://doi.org/10.1016/j.stem.2012.04.027
Medvedev SP, Grigor’eva EV, Shevchenko AI, Malakhova AA, Dementyeva EV, Shilov AA, Pokushalov EA, Zaidman AM, Aleksandrova MA, Plotnikov EY, Sukhikh GT, Zakian SM (2011) Human induced pluripotent stem cells derived from fetal neural stem cells successfully undergo directed differentiation into cartilage. Stem Cells Dev 20:1099–1112. https://doi.org/10.1089/scd.2010.0249
Mena MA, Casarejos MJ, Bonin A, Ramos JA, García Yébenes J (1995) Effects of dibutyryl cyclic AMP and retinoic acid on the differentiation of dopamine neurons: prevention of cell death by dibutyryl cyclic AMP. J Neurochem 65:2612–2620. https://doi.org/10.1046/j.1471-4159.1995.65062612.x
Morozova KN, Suldina LA, Malankhanova TB, Grigoreva EV, Zakian SM, Kiseleva E, Malakhova AA (2018) Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells. PLoS ONE 13:e0204735. https://doi.org/10.1371/journal.pone.0204735
Nekrasov ED, Vigont VA, Klyushnikov SA, Lebedeva OS, Vassina EM, Bogomazova AN, Chestkov IV, Semashko TA, Kiseleva E, Suldina LA, Bobrovsky PA, Zimina OA, Ryazantseva MA, Skopin AY, Illarioshkin SN, Kaznacheyeva EV, Lagarkova MA, Kiselev SL (2016) Manifestation of Huntington’s disease pathology in human induced pluripotent stem cell-derived neurons. Mol Neurodegene 11:1–15. https://doi.org/10.1186/s13024-016-0092-5
Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412. https://doi.org/10.1038/nmeth.1591
Sharma S, Hansen J, Notte M (1990) Effects of NGF and dibutyryl cAMP on neuronal differentiation of embryonal carcinoma cells. Int J Dev Neurosci 8:33–45. https://doi.org/10.1038/nmeth.1591
Skogh C, Parmar M, Campbell K (2003) The differentiation potential of precursor cells from the mouse lateral ganglionic eminence is restricted by in vitro expansion. Neuroscience 120:379–385. https://doi.org/10.1016/s0306-4522(03)00427-5
Tojima T, Kobayashi S, Ito E (2003) Dual role of cyclic AMP-dependent protein kinase in neuritogenesis and synaptogenesis during neuronal differentiation. J Neurosci Res 74:829–837. https://doi.org/10.1002/jnr.10754
Wang H, Luo X, Leighton J (2015) Extracellular matrix and integrins in embryonic stem cell differentiation Biochem Insights 8:15–21. https://doi.org/10.4137/BCI.S30377
Wilson PG, Stice SS (2006) Development and differentiation of neural rosettes derived from human embryonic stem cells. Stem Cell Rev 2:67–77. https://doi.org/10.1385/SCR:2:1:67
Xu X, Tay Y, Sim B, Yoon SI, Huang Y, Ooi J, Utami KH, Ziaei A, Ng B, Radulescu C, Low D, Ng AYJ, Loh M, Venkatesh B, Ginhoux F, Augustine GJ, Pouladi MA (2017) Reversal of phenotypic abnormalities by CRISPR/Cas9-mediated gene correction in Huntington disease patient-derived induced pluripotent stem cells. Stem Cell Reports 8:619–633. https://doi.org/10.1016/j.stemcr.2017.01.022
Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324:797–802. https://doi.org/10.1126/science.1172482
Zahir T, Chen YF, MacDonald JF, Leipzig N, Tator CH, Shoichet MS (2009) Neural stem/progenitor cells differentiate in vitro to neurons by the combined action of dibutyryl cAMP and interferon-gamma. Stem Cells Dev 18:1423–1432. https://doi.org/10.1089/scd.2008.0412
Zuccato C, Cattaneo E (2007) Role of brain-derived neurotrophic factor in Huntington’s disease. Prog Neurobiol 81:294–330. https://doi.org/10.1016/j.pneurobio.2007.01.003
Acknowledgements
The study was supported by the Russian Science Foundation (Project No 16-15-10128). The microscopy was conducted at the Joint Access Center for Microscopy of Biological Objects with the Siberian Branch of the Russian Academy of Sciences. We thank S. I. Bayborodin for the technical assistance. Teratoma test is implemented using the equipment of the Center for Genetic Resources of Laboratory Animals at ICG SB RAS, supported by the Ministry of Education and Science of Russia (Unique identifier of the project RFMEFI62117X0015). Biology material was provided by Research Institute of Medical Genetics, Tomsk NRMC.
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Suren M. Zakian, Elena V. Grigor’eva, Igor N. Lebedev and Anastasia A. Malakhova conceived and designed the experiments. Elena V. Grigor’eva and Aizhan Surumbayeva reprogrammed and differentiated the cells. Elena V. Grigor’eva, Sophia V. Pavlova and Anastasia A. Malakhova performed immunofluorescent analysis and cell analysis by flow cytometry. Tuyana B. Malankhanova performed transfection experiments, RNA isolation, RT-PCR and qRT-PCR. Julia M. Minina performed cytogenetic analysis. Elena A. Kizilova performed teratoma assay. Lyubov A. Suldina, Ksenia N. Morozova and Elena Kiseleva performed electron microscopy. Evgeny D. Sorokoumov performed electrophysiology. Elena V. Grigor’eva, Tuyana B. Malankhanova and Anastasia A. Malakhova wrote the main manuscript text. All authors read and approved the final manuscript.
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Grigor’eva, E.V., Malankhanova, T.B., Surumbayeva, A. et al. Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells. Cytotechnology 72, 649–663 (2020). https://doi.org/10.1007/s10616-020-00406-7
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DOI: https://doi.org/10.1007/s10616-020-00406-7