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Materials

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Neuroscience

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

Published: October 2nd, 2021

DOI:

10.3791/62095

1Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, UK, 2Department of Physiology and Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland, 3Regenerative Medicine Laboratory, School of Clinical Sciences, University of Bristol, Bristol, UK
* These authors contributed equally

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.

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.

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 death1,2,3. As the dopaminergic neurons are the degenera....

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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 con.......

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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 (

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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.......

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This work was funded by a Parkinson's UK project grant (G-1402) and studentship. The authors gratefully acknowledge the Wolfson Bioimaging Facility for their support and assistance in this work.

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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

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