An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons
While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure–function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.
KeywordsMouse embryonic stem cells Cerebellar Purkinje neurons Cerebellar granule neurons Co-culture
We thank Dr. Chengyu Lui (NHLBI Transgenic Core) for providing the mouse embryonic stem cells.
This study was funded by the National Heart, Lung, and Blood Institute Intramural Research Program at the National Institutes of Health.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
All animal care and experiments performed in this study were approved by the Institutional Animal Care and Use Committee of the National Heart, Lung, and Blood Institute in accordance with the National Institute of Health guidelines. This article does not contain any studies with human participants performed by any of the authors.
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