Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

Summary

Here, we present a method for reproducible generation of ventral midbrain patterned astrocytes from hiPSCs and protocols for their characterization to assess phenotype and function.

Abstract

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required.

We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

Introduction

Parkinson's disease affects 2%-3% of people over 65 years of age, making it the most prevalent neurodegenerative movement disorder¹. It is caused by degeneration of ventral midbrain dopamine neurons within the substantia nigra, resulting in debilitating motor symptoms, as well as frequent cognitive and psychiatric issues². Parkinson's pathology is typified by aggregates of the protein, α-synuclein, which are toxic to neurons and result in their dysfunction and death^1,2,3. As the dopaminergic neurons are the degenera....

Protocol

1. Human hiPSC line thawing, maintenance, and cryopreservation

1. For coating hiPSC culture plates, dilute vitronectin to 5 µg/mL (1:100) in PBS at 1 mL per 10 cm² cell culture plate surface area. Leave for 1 h at room temperature.
2. Remove vitronectin and proceed immediately to adding hiPSCs/media to the culture plate.
   NOTE When removing vitronectin from the plate, it is crucial that the culture surface is not allowed to dry out.
3. To thaw hiPSCs, remove cryovials containing hiPSCs from liquid nitrogen and place in a 37 °C water bath until the contents have completely thawed.
4. Prepare 9 mL of prewarme....

Results

Differentiation methodology and progression
Here we present the details of both the methods employed for the generation of vmAstros and the protocols used for their subsequent phenotypic characterization. The method for generation of vmAstros is made up of several distinct differentiation stages, which can be monitored by microscopy and identifying distinct morphological characteristics (Figure 1A-F). A feeder-free hiPSC culture (

Discussion

This method for the generation of vmAstros from hiPSCs is highly efficient, generating pure cultures of vmAstros, and being reproducible for the generation of vmAstros from different hiPSC lines. This protocol was developed around the recapitulation of the developmental events required in the embryo to correctly pattern the developing midbrain and generate astrocytes and comprises three defined stages: 1) neural ventral midbrain induction to generate vmNPCs, 2) generation and expansion of vmAPCs, and finally 3) maturatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
0.2M Tris-Cl (pH 8.5)	n/a	n/a	Made up from Tris base and plus HCl
0.5M EDTA, PH 8	ThermoFisher	15575-020	1:1000 in D-PBS to 0.5 mM final
1,4-diazabicylo[2.2.2]octane (DABCO)	Sigma	D27802-	25 mg/mL in Mowiol mounting solution
13 mm coverslips	VWR	631-0149
2-Mercaptoethanol (50 mM)	ThermoFisher	31350010
Accutase	ThermoFisher	13151014
Advanced DMEM/F12	ThermoFisher	12634010	Has 1x NEAA but we add to final concentration of 2x (0.2 mM)
Ascorbic acid	Sigma	A5960	200 mM stock, 1:1000 to 200 µM final
B27 Supplement	ThermoFisher	17504-044	50x stock
BSA	Sigma	5470
Cell freezing media	Sigma	C2874	Cryostor CS10
Cell freezing vessel	Nalgene	5100-0001
CHIR99021	Axon Medchem	1386	0.8 mM stock, 1:1000 dilution to 0.8 µM final
Cryovials	Sigma	CLS430487
DAPI	Sigma	D9542	1 mg/mL, 1:10,000 to 100ng/mL final (in PBS)
DMEM/F12 + Glutamax	ThermoFisher	10565018
Dulbeccos-PBS (D-PBS without Mg or Ca)	ThermoFisher	14190144	pH 7.2
E8 Flex medium kit	ThermoFisher	A2858501
Formaldehyde (36% solution)	Sigma	47608
Geltrex	ThermoFisher	A1413302	1:100 or 1:400 in ice-cold DMEM/F12
Glutamax	ThermoFisher	35050038	2 mM stock (1:200 in N2B27, 1:100 in ASTRO media to 20 µM final)
Glycerol	Sigma	G5516
Human BDNF	Peprotech	450-02	20 µg/mL stock, 1:1000 to 20 ng/mL final
Human BMP4	Peprotech	120-05	20 µg/mL stock, 1:1000 to 20 ng/mL final
Human EGF	Peprotech	AF-100-15	20 µg/mL stock, 1:1000 to 20 ng/mL final
Human GDNF	Peprotech	450-10	20 µg/mL stock, 1:1000 to 20 ng/mL final
Human insulin solution	Sigma	I9278	10 mg/mL stock, 1:2000 to 5 µg/mL final
Human LIF	Peprotech	300-05	20 µg/mL stock, 1:1000 to 20 ng/mL final
IL-6 ELISA kit	Biotechne	DY206
Isopropanol	Sigma	I9516-4L	For filling Mr Frosty cryostorage vessel
LDN193189	Sigma	SML0559	100 µM stock, 1:10,000 dilution to 10 nM final
Mowiol 40-88	Sigma	324590
N2 Supplement	ThermoFisher	17502048	100x stock
NEAA	ThermoFisher	11140035	10 mM stock, 1:100 to 0.1 mM final
Neurobasal media	ThermoFisher	21103049
Normal Goat serum	Vector Labs	S-1000-20
Revitacell	ThermoFisher	A2644501	100x stock, 1:100 to 1x final
SB431542	Tocris	1614	10 mM stock, 1:1000 dilution to 10 µM final
SHH-C24ii	Biotechne	1845-SH-025	200 µg/mL stock, 1:1000 to 200 ng/mL final
Tris-HCl	Sigma	PHG0002
Triton-X	Sigma	X100
Tween-20	Sigma	P7949
Vitronectin	ThermoFisher	A14700	1:50 in D-PBS
Antibodies for immunocytochemistry	Company	Catalogue Number	Host species
Antibody against S100b	Sigma	SAB4200671	Mouse; 1:200
Antibody against FOXA2	SCBT	NB600501	Mouse; 1:50
Antibody against LMX1A	ProSci	7087	Rabbit; 1:300
Antibody against LMX1A	Millipore	AB10533	Rabbit; 1:2000
Antibody against LMX1B	Proteintech	18278-1-AP	Rabbit; 1:300
Antibody against GLAST	Proteintech	20785-1-AP	Rabbit; 1:300
Antibody against GFAP	Dako	Z0334	Rabbit; 1:400
Antibody against CD49f	Proteintech	27189-1-AP	Rabbit; 1:100
Antibody against MSI1	Abcam	ab52865	Rabbit; 1:400
Alexa Fluor 488 Goat Anti-Rabbit	ThermoFisher	A32731	Goat; 1:500
Alexa Fluor 488 Goat Anti-Mouse	ThermoFisher	A32723	Goat; 1:500
Alexa Fluor 568 Goat Anti-Rabbit	ThermoFisher	A11036	Goat; 1:500
Alexa Fluor 488 Goat Anti-Mouse	ThermoFisher	A11031	Goat; 1:500