Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Summary

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Abstract

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

Introduction

In both mice and humans, roughly half of all cortical inhibitory interneurons (CIns) originate within a transient subcortical structure known as the medial ganglionic eminence (MGE), where the neuroepithelial progenitors of CIns and other neuronal and glial subgroups express the transcription factor Nkx2.1^1,2. CIn subgroups or subtypes are defined by intersecting morphological, neurochemical, electrophysiological, and connectivity characteristics^3,4. The MGE-derived CIns can be grouped into mostly non-overlapping subgroups based on their expression of ....

Protocol

NOTE: The dual reporter mESC line described in this protocol is available upon request (sande@mail.med.upenn.edu).

1. Media Preparation

NOTE: Warm all media to 37 °C before use in cell culture.

1. Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
   1. Add 50 mL fetal bovine serum (FBS) to 449 mL of Dulbecco's Modified Eagle's Medium (DMEM), and filter through a 500 mL 0.22 µm pore filter unit.
   2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C for up to 1 month or aliquot and store at ≤ -20 °C.....

Results

The protocol described in this paper is a modified version of our published protocols^15,16,17 and has been optimized for use with our Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC line. By adding the Wnt inhibitor XAV-939 from DD0-5, combined with re-plating at DD8, we achieve robust Nkx2.1 induction, wherein upwards of 50% of all DAPI+ nuclei in culture are also Nkx2.1 expressing (

Discussion

While this method is highly effective at patterning J1-derived mESCs (SCRC-1010), we have experienced variable success with other mESC lines and clonal isolates. For instance, Foxg1::venus mESCs (EB3-derived; Danjo et al.¹³) respond poorly to this protocol and Foxg1 induction by DD12 is typically on the order of 1-2%. For reasons that we do not fully understand, another Nkx2.1::mCherry:Lhx6::GFP dual reporter clone (termed JQ59) that was isolated simultaneously as the line described in th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bottle-top vacuum filter system	Corning	CLS430769
Test Tube with Cell Strainer Snap Cap	ThermoFisher	Corning 352235
Mouse embryonic fibroblasts (CF-1 MEF IRR 7M)	MTI-Globalstem	GSC-6101G	1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: 29,35
FBS	Atlanta Biologicals	S11150H
Primocin	Invivogen	Ant-pm-2	Also known as antimicrobial agent. Do not filter with base media -- add after filtration. References: 9,11,36,37
N2 supplement-B	Stemcell Technologies	7156	Do not filter with base media -- add after filtration
Glutamax (100x)	ThermoFisher	35050061	Also known as L-alanine-L-glutamine. References: 9,11,38,39
KnockOut Serum Replacement (KSR)	ThermoFisher	10828028	Also known as serum-free medium supplement. References: 9,11
L-glutamine (100x)	ThermoFisher	25030081
MEM-NEAA (100x)	ThermoFisher	11140050
2-Mercaptoethanol	ThermoFisher	21985023
KnockOut DMEM	ThermoFisher	10829018	Also known as non-glutamine containing DMEM. References: 9,11
Hyclone FBS	VWR	82013-578	Also known as stem cell grade FBS. References: 9,11
Tissue culture treated dish (10cm)	BD Falcon	353003
Non-adherent sterile petri dish (10cm)	VWR	25384-342
Leukemia inhibitory factor (mLIF)	Chemicon	ESG1107	Do not freeze, store at 4'C. References: 9,11
DMEM/F12	ThermoFisher	11330032
0.1% Gelatin Solution	ATCC	ATCC PCS-999-027
Laminin	Sigma	L2020
Poly-L-lysine	Sigma	P6282
Trypsin-EDTA (0.05%)	ThermoFisher	25300054
Accutase	ThermoFisher	A1110501	Also known as non-trypsin containing cell dissociation reagent. References: 9,11
RQ1 RNase-Free DNase	Promega	M610A
LDN-193189	Stemgent	04-0074	Resuspend in DMSO and store at -80'C in single use aliquots
XAV939	Stemgent	04-0046	Resuspend in DMSO and store at -80'C in single use aliquots
rhFGF-2	R&D Systems	233-FB	Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
rhIGF-2	R&D Systems	291-G1	Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
ROCK inhibitor (Y-27632)	Tocris	1254	Resuspend in DMSO and store at -80'C in single use aliquots
Smoothened agonist (SAG)	Millipore	566660-1MG	Resuspend in H20 and store at -80'C in single use aliquots
rm Sonic Hedgehog/SHH	R&D Systems	464-SH-025	Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
PKCζ Pseudosubstrate Inhibitor, Myristoylated	EMD Millipore	539624	Resuspend in H20 and store at -80'C in single use aliquots