title: Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMPreporter line.
abstract: In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32WT/eGFP-AMP-LacZ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia.
authors.forename: C P J
authors.initials: CPJ
authors.lastname: Hunt
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia.
authors.forename: C W
authors.initials: CW
authors.lastname: Pouton
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia. Electronic address: john.haynes@monash.edu.
authors.forename: J M
authors.initials: JM
authors.lastname: Haynes
mesh_terms: D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology*; D053823:Hedgehog Proteins / Q000502:physiology; D000066449:Human Embryonic Stem Cells / Q000502:physiology*; D006801:Humans; D009474:Neurons / Q000502:physiology*; D060449:Wnt Signaling Pathway / Q000502:physiology
chemical_list: D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D053823:Hedgehog Proteins; C497928:PPP1R1B protein, human; C509239:SHH protein, human
keywords: Development; Gene editing; Human embryonic stem cells; Medium spiny neurons; Neural differentiation; Pharmacology