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Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells

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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

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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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Correspondence to Elena V. Grigor’eva.

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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

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