Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Summary

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Abstract

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

Introduction

Postganglionic sympathetic neurons (symNs) belong to the autonomic nervous system (ANS) and have multiple important roles in responding and regulating homeostasis of the body independent of consciousness. For example, stress stimulates symNs and evokes the fight-or-flight response that leads to an increase in heart rate, blood pressure, and sweating. SymNs are affected in multiple human disorders due to genetics, toxicity/injury, or as companions to other diseases. An example of a genetic neuropathy is the childhood disorder Familial Dysautonomia (FD), where a severe dysregulation of symNs causes dysautonomic crisis, evident by sweating, blotching of the skin, vomitin....

Protocol

NOTE: The H9 PHOX2B:GFP reporter line was provided by Oh et al.¹⁹. Some qPCR primers used in this paper were obtained from OriGene Technologies, while a few sequences are obtained from Frith et al.^20,30.

1. Set-up for dish coating, media preparation, and hPSC maintenance

1. Dish coating
   1. Vitronectin (VTN) coating
      1. Place vials of VTN in a 37 °C water bath until fully thawed, then mix thoroughly.
      2. For a 100 mm Petri dish, mix 7 mL of 1x phosphate buffered saline (PBS) with 0.5 mg/mL VTN, add VTN ....

Results

In this protocol, we give instructions on how to generate symNs from hPSCs. The culture conditions demonstrated here were improved from an earlier published protocol^23,24 (Figure 1A) to feeder-free and chemically defined conditions (Figure 1B). Two options are provided, one where symNs are made within 20 days, and another where the NCCs can be expanded for 2 weeks to generate more cells that can then be .......

Discussion

We recently published two reviews, one discussing the use of hPSC-derived symNs for disease modeling³¹ as well as an in-depth comparison of available differentiation protocols²². Thus, here we focus on troubleshooting the current protocol to help the interested researcher succeed in making symNs. During the entire differentiation process, in order to gain consistent data as well as healthy differentiated cells, contamination at all stages should be carefully controlled. In .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mm cell culture dishes	Falcon	353003
15 mL conical tissue culture tubes	VWR/Corning	89039-664
24-well tissue culture plates	Falcon	353047
24-well ultra-low-attachment plates	Corning	07 200 601 and 07 200 602
5% CO2/20% O2 tissue culture incubator	Thermo Fisher/Life Technologies	Heracell VIOS 160i
50 ml conical tissue culture tubes	VWR/Corning	89039-656
6-well tissue culture plates	Costar	3516
Accutase	Innovation Cell Technologies	AT104500	Cell dissociation solution
Anti-AP2a antibody	Abcam	ab108311	Host: Rabbit; 1:400 dilution
Anti-Ascl1 antibody	BD Pharmingen	556604	Host: Mouse IgG1; 1:200 dilution
Anti-CD49D antibody	BioLegend	304313	Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
Anti-CD49D antibody (isotype)	BioLegend	400125	Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
DAPI dye	Sigma	D9542	1:1000 dilution
Anti-DBH antibody	Immunostar	22806	Host: Rabbit; 1:500 dilution
Anti-GFP antibody	Abcam	ab13970	Host: Chicken; 1:1000 dilution
Anti-HOXC9 antibody	Abcam	ab50839	Host: Mouse; 1:100 dilution
Anti-NET1 antibody	Mab	NET17-1	Host: Mouse; 1:1000 dilution
Anti-PRPH antibody	Santa Cruz Biotechnology	SC-377093/H0112	Host: Mouse IgG2a; 1:200 dilution
Anti-SOX10 antibody	Santa Cruz Biotechnology	sc-365692	Host: Mouse IgG1; 1:100 dilution
Anti-TH antibody	Pel-Freez	P40101- 150	Host: Rabbit; 1:500 dilution
Ascorbic acid	Sigma	A8960-5G	Stock concentration: 100 mM
B27 supplement	Thermo Fisher/Life Technologies	12587-010	Stock concentration: 50x
BDNF	R&D Systems	248-BD	Stock concentration: 10 μg/mL
BMP4	R&D Systems	314-BP	Stock concentration: 6 mM
Cell counter	Thermo Fisher/Life Technologies	Countess II
Cell counting chamber slides	Invitrogen	C10312
Centrifuge	Eppendorf	57021&5424R
CHIR99021	R&D Systems	4423	Stock concentration: 6 mM
Cryo-vial	Thermo Fisher/Life Technologies	375353
dbcAMP	Sigma	D0627	Stock concentration: 100 mM
DMEM	Thermo Fisher/Life Technologies	10829-018	Stock concentration: 1x
DMEM/F12	Thermo Fisher/Life Technologies	11330-057	Stock concentration: 1x
DMSO	Thermo Fisher/Life Technologies	BP231-100
E6 medium	gibco	A15165-01
E8 medium	gibco	A15169-01	Stock concentration: 1x
E8 supplement	gibco	A15171-01	Stock concentration: 50x
EDTA	Sigma	ED2SS	Stock concentration: 0.5 M
Electrophysiology plates (AXION cytoview MEA96)	Axion BioSystems	M768-tMEA-96W
FACS machine	Beckman Coulter	CytoFLEX (for FACS)
FACS machine	Beckman Coulter	MoFlo Astrios EQ (for sorting)
FACS tubes (blue filter cap)	Falcon	352235
FACS tubes (white cap)	Falcon	352063
Fetal bovine serum (FBS)	Atlanta Biologicals	S11150
GDNF	PeproTech	450	Stock concentration: 10 μg/mL
Geltrex	Invitrogen	A1413202	Basement membrane matrix; Stock concentration: 100x
hPSCs	Thomson et al., (1998)	WA09
hPSCs	Oh et al. (2016)	H9-PHOX2B::eGFP
Human fibronectin (FN)	VWR/Corning	47743-654	Stock concentration: 1 mg/mL
L-glutamine	Thermo Fisher/Gibco	25030-081	Stock concentration: 200 mM
LN tank	Custom Biogenic Systems	V-1500AB
MEA reader	Axion BioSystems	Maestro Pro
Mouse laminin I (LM)	R&D Systems	3400-010-01	Stock concentration: 1 mg/mL
N2 supplement	Thermo Fisher/Life Technologies	17502-048	Stock concentration: 100x
Neurobasal medium	gibco	21103-049	Stock concentration: 1x
NGF	PeproTech	450-01	Stock concentration: 25 μg/mL
Phosphate-buffered saline (PBS)	Gibco	14190-136	Stock concentration: 1x
Poly-L-ornithine hydrobromide (PO)	Sigma	P3655	Stock concentration: 15 mg/mL
Primocin (antibiotics)	InvivoGen	ANTPM1	Stock concentration: 50 mg/mL
qPCR machine	Bio-Rad Laboratories	C1000 Touch
qPCR plates	Bio-Rad Laboratories	HSP9601
recombinant FGF2	R&D Systems	233-FB/CF	Stock concentration: 10 μg/mL
Retinoic acid	Sigma	R2625	Stock concentration: 1 mM
SB431542	Tocris/R&D Systems	1614	Stock concentration: 10 mM
Trypan blue	Corning	MT-25-900-CI
Vitronectin (VTN)	Thermo Fisher/Life Technologies	A14700	Stock concentration: 0.5 mg/mL
Water bath	VWR/Corning	706308
Y27632	R&D Systems	1254	Stock concentration: 10 mM