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An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons

  • Christopher J. Alexander
  • John A. HammerEmail author
Original Paper

Abstract

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.

Keywords

Mouse embryonic stem cells Cerebellar Purkinje neurons Cerebellar granule neurons Co-culture 

Notes

Acknowledgements

We thank Dr. Chengyu Lui (NHLBI Transgenic Core) for providing the mouse embryonic stem cells.

Funding

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.

Ethical Approval

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.

Supplementary material

12311_2019_1007_Fig8_ESM.png (3 mb)
Figure S1

Generation of PN precursors and GN precursors. Shown is a representative example of a precursor PN culture 10 days after initiation from NP cells that was stained for TubJ to mark all neurons (A1) and Neph3 to mark PN precursors (A2). The overlaid image in shown in A3. Also shown is a representative example of a precursor GN culture 12 days after initiation from NP cells that was stained for TubJ to mark all neurons (B1) and NeuN to mark GN precursors (B2). The overlaid image in shown in B3. Mag bars: 100 μm (A3 and B3). (PNG 3039 kb)

12311_2019_1007_MOESM1_ESM.tif (4 mb)
High Resolution Image (TIF 4082 kb)
12311_2019_1007_Fig9_ESM.png (144 kb)
Figure S2

A small number of GN precursors develop into Calbindin D28K-positive cells. Shown is a representative example of a Calbindin D28K-positive cell present when GN precursor cells are cultured for 14 days in PN Basal Growth Medium. Mag bar: 20 μm (PNG 143 kb)

12311_2019_1007_MOESM2_ESM.tif (192 kb)
High Resolution Image (TIF 192 kb)
12311_2019_1007_Fig10_ESM.png (642 kb)
Figure S3

An optimized extracellular substrate creates thinner cultures suitable for high-resolution microscopy. Shown is a representative example of a GN/PN precursor co-culture grown for 21 days on gelatin (the standard surface) (A), or on a combination of Geltrex, poly-l-lysine (PLL) and poly-l-ornithine (PLO) (B). The latter substrate results in thinner cultures more suitable for high-resolution microscopy. Mag bars: 100 μm (A and B). (PNG 642 kb)

12311_2019_1007_MOESM3_ESM.tif (837 kb)
High Resolution Image (TIF 836 kb)
12311_2019_1007_Fig11_ESM.png (75 kb)
Figure S4

Yield of developed PNs per dish. The co-culture of GN/PN precursors yields a similar number of developed PNs per dish as primary culture. Cells were cultured on Cellvis 35 mm #1.5 glass-bottomed dishes having a total growth area of 154 mm2. A total of 3 independent experiments for each condition were analyzed. Error bars represent the standard deviation; n.s., not significant. (PNG 74 kb)

12311_2019_1007_MOESM4_ESM.tif (237 kb)
High Resolution Image (TIF 237 kb)
12311_2019_1007_Fig12_ESM.png (3.4 mb)
Figure S5

Primary PNs express multiple proteins used previously to mark PNs. Shown are representative Day-21 primary PNs that were double-stained for the PN-specific protein Calbindin D28K in red and the following proteins known to be expressed by PNs in green: PSD93 (A1-A4), PLCβ4 (B1-B4), myosin IIB-B2 (C1-C4), IP3R1 (D1-D4), and IRBIT (E1-E4). Each sample is presented as a lower magnification, overlaid image in the first panel, and higher magnification split and overlaid images of the boxed region in the second, third and fourth panels, respectively. Mag bars: 20 μm (A1-E1) and 5 μm (A4-E4). (PNG 3523 kb)

12311_2019_1007_MOESM5_ESM.tif (4.8 mb)
High Resolution Image (TIF 4907 kb)
12311_2019_1007_Fig13_ESM.png (816 kb)
Figure S6

mESC-derived PNs express exogenous proteins off the PN-specific promoter Pcp2/L7. Shown are representative examples of a mESC-derived PN (A1, inset of boxed region in A2) and a primary PN (B1, inset of boxed region in B2) that were transfected biolistically with a Pcp2/L7 plasmid [11] driving the expression of free GFP. Mag bars: 20 μm (A1, B1) and 5 μm (A2, B2). (PNG 816 kb)

12311_2019_1007_MOESM6_ESM.tif (1.1 mb)
High Resolution Image (TIF 1112 kb)

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Cell and Developmental Biology Center, National Heart, Lung, and Blood InstituteNational Institutes of HealthBethesdaUSA

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