Differentiation of human induced pluripotent stem cells into cortical neural stem cells

Alexandra Neaverson; Malin H L Andersson; Osama A Arshad; Luke Foulser; Mary Goodwin-Trotman; Adam Hunter; Ben Newman; Minal Patel; Charlotte Roth; Tristan Thwaites; Helena Kilpinen; Matthew E Hurles; Andrew Day; Sebastian S Gerety

doi:10.3389/fcell.2022.1023340

Differentiation of human induced pluripotent stem cells into cortical neural stem cells

Front Cell Dev Biol. 2023 Jan 5:10:1023340. doi: 10.3389/fcell.2022.1023340. eCollection 2022.

Authors

Alexandra Neaverson^{1

2}, Malin H L Andersson¹, Osama A Arshad¹, Luke Foulser^{1

2}, Mary Goodwin-Trotman¹, Adam Hunter¹, Ben Newman¹, Minal Patel¹, Charlotte Roth³, Tristan Thwaites¹, Helena Kilpinen^{1

2

3

4

5}, Matthew E Hurles^{1

2}, Andrew Day¹, Sebastian S Gerety^{1

2}

Affiliations

¹ Wellcome Sanger Institute, Cambridge, United Kingdom.
² Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom.
³ UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.
⁴ Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
⁵ Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.

Abstract

Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Keywords: IPSC; RNA-seq; developmental disorders; in vitro disease modelling; neural differentiation; neural stem cell; protocol.

Abstract

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