Derivation of enkephalinergic medium spiny neurons from mouse embryonic stem cells
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Date
29/06/2015Author
Vatanashevanopakorn, Chinnavuth
Metadata
Abstract
Medium spiny neurons (MSNs) play an important role in locomotion.
Counterbalance between two MSN subtypes, enkephalin-positive and substance P-positive
MSNs, is crucial for maintaining normal movement. Preferential
degeneration of enkephalinergic MSNs in early stage Huntington’s disease (HD)
contributes to abnormal involuntary movement called chorea. The reasons for this
selective vulnerability are unknown. In vitro differentiation of pluripotent stem cells
(PSCs) to neuronal cells is considered a potential approach for modelling
neurodegenerative disorders including HD. Generation of PSC-derived
enkephalinergic MSNs would be an ideal tool for dissecting their preferential
degeneration. However, an enkephalinergic phenotype has never been reported in
PSC-derived MSNs. We, therefore, have generated a mouse embryonic stem cell
(mESC) reporter line that expresses enhanced yellow fluorescent protein (EYFP)
when the cells are committed to an enkephalinergic fate. Characterisation of this
mESC line via chimaera generation showed that all EYFP-positive cells were also
enkephalin-positive. We have then optimised an enkephalinergic neuronal
differentiation protocol using this ESC line. Interestingly, we found that a
combination of Wnt inhibitor Dickkopf-related protein 1 (DKK1), sonic hedgehog
(Shh) and brain-derived neurotrophic factor (BDNF), commonly used in addition to
basal medium for deriving MSNs from PSCs, had a detrimental effect on enkephalin
expression. Absence of these three factors, surprisingly, did not reduce the potential
of ESCs to become MSNs nor did it affect the electrophysiological properties of
ESC-derived MSNs. Further investigation revealed that Pre-pro-enkephalin is down-regulated
in the presence of exogenous DKK1 and/or Shh but not in the presence of
BDNF. We, therefore, propose that addition of exogenous DKK1 and Shh is
unfavourable to derive enkephalinergic MSNs from mouse ESCs. These findings
could be used to derive enkephalinergic MSNs in vitro allowing the disease in a dish
approach for HD modelling.