Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells.
Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling.
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom.
Bilada
B
Bilican
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
Matthew R
MR
Livesey
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
Ghazal
G
Haghi
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
Jing
J
Qiu
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom.
Karen
K
Burr
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom.
Rick
R
Siller
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
Giles E
GE
Hardingham
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
David J A
DJ
Wyllie
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom.
Siddharthan
S
Chandran
D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013569:Synapses / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D010100:Oxygen