Robust neuronal differentiation of human embryonic stem cells for neurotoxicology

Athina Samara; Martin Falck; Mari Spildrejorde; Magnus Leithaug; Ganesh Acharya; Robert Lyle; Ragnhild Eskeland

doi:10.1016/j.xpro.2022.101533

Robust neuronal differentiation of human embryonic stem cells for neurotoxicology

STAR Protoc. 2022 Sep 16;3(3):101533. doi: 10.1016/j.xpro.2022.101533.

Authors

Athina Samara¹, Martin Falck², Mari Spildrejorde³, Magnus Leithaug⁴, Ganesh Acharya⁵, Robert Lyle⁶, Ragnhild Eskeland⁷

Affiliations

¹ Division of Clinical Paediatrics, Department of Women's and Children's Health, Karolinska Institutet, 17177 Stockholm, Sweden; Astrid Lindgren Children's Hospital Karolinska University Hospital, 17177 Stockholm, Sweden. Electronic address: athina.samara@ki.se.
² Department of Biosciences, University of Oslo, Blindern, PO Box 1066, 0316 Oslo, Norway; PharmaTox Strategic Research Initiative, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway.
³ PharmaTox Strategic Research Initiative, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway; Department of Medical Genetics and Norwegian Sequencing Centre, Oslo University Hospital, Kirkeveien 166, 0450 Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0450 Oslo, Norway.
⁴ PharmaTox Strategic Research Initiative, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway; Department of Medical Genetics and Norwegian Sequencing Centre, Oslo University Hospital, Kirkeveien 166, 0450 Oslo, Norway.
⁵ Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Alfred Nobels Allé 8, 14152 Stockholm, Sweden; Center for Fetal Medicine, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden.
⁶ PharmaTox Strategic Research Initiative, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway; Department of Medical Genetics and Norwegian Sequencing Centre, Oslo University Hospital, Kirkeveien 166, 0450 Oslo, Norway; Centre for Fertility and Health, Norwegian Institute of Public Health, PO 222 Skøyen, 0213 Oslo, Norway.
⁷ PharmaTox Strategic Research Initiative, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway; Institute of Basic Medical Sciences, Department of Molecular Medicine, Faculty of Medicine, University of Oslo, Blindern, PO Box 1112, 0317 Oslo, Norway. Electronic address: ragnhild.eskeland@medisin.uio.no.

Abstract

Here, we describe a protocol for rapid neuronal differentiation from human embryonic stem cells (hESCs) toward a heterogenous population of telencephalic progenitors, immature and mature neurons, for drug-screening and early-brain differentiation studies. hESC neuronal differentiation depends on adhesion and minimal cell-passaging to avert monolayer cross-connectivity rupture. In this protocol, we detail optimized cell-seeding densities and coating conditions with high cell viability suitable for neurotoxicology and high-resolution single-cell omics studies. Daily media changes reduce compound instability and degradation for optimal screening. For complete details on the use and execution of this protocol, please refer to Samara et al. (2022).

Keywords: Cell Differentiation; Cell culture; Developmental biology; Gene Expression; Neuroscience; Stem Cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation / physiology
Cell Survival
Embryonic Stem Cells
Human Embryonic Stem Cells*
Humans
Neurons