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Abstract

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Materials

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Neuroscience

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Published: July 7th, 2023

DOI:

10.3791/65570

1Ksilink

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Parkinson's disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, α-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

A variety of cell biological processes are disturbed in Parkinson's disease (PD). For example, mitochondrial dysfunction, oxidative stress, protein degradation defects, disruption of vesicular trafficking and endolysosomal function have been associated with midbrain dopaminergic (mDA) neuron loss, are commonly observed in PD1. Therefore, PD appears to involve multiple disease mechanisms that can interact with and worsen each other. One useful way to investigate this mechanistic interplay is the creation of a comprehensive phenotypic fingerprint or profile of midbrain dopaminergic (mDA) neurons.

Phenotypic profili....

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1. Preparation of medium and plates for neuron seeding (Day 1)

  1. To prepare the plates for neuron seeding on Day-1, warm Laminin to room temperature (RT) just before use. Prepare the Laminin solution by diluting the Laminin stock solution (0.1 mg/mL) 1/10 in cold PBS+/+ (with Ca2+ and Mg2+).
    NOTE: All reagents are listed in the Table of Materials. The compositions of solutions and buffers are described in Tables 1-4.
  2. Then,.......

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Phenotypic profiling in mDA neurons is an efficient way to quantify multiple aspects of cellular biology and their changes during the experimental modulation. To exemplify this methodology, this study made use of cryopreserved LRRK2 G2019S and healthy donor mDA neurons. These neurons have been differentiated for approximately 37 days, are post-mitotic and express neuronal markers (TUBB3 and MAP2) and dopaminergic neuron markers, including tyrosine hydroxylase (TH) in combination with FOXA2, while the glial marker Glial F.......

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Phenotypic profiling is a technique to measure a large number of phenotypes in cells by applying fluorescent stainings, microscopy, and image analysis3. Phenotypic profiles can be obtained and compared across cell lines or other experimental conditions to understand complex changes in cellular biology that might go unnoticed when using a single readout. Here we describe the application of phenotypic profiling to human iPSC-derived mDA neurons, a cell type frequently used to model PD cellular biolo.......

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The authors would like to thank all colleagues at Ksilink for their valuable help and discussions that lead to the design of the presented protocol.

....

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Name Company Catalog Number Comments
Anti- chicken – Alexa 647 Jackson ImmunoRearch 703-605-155 Immunofluorescence
Anaconda https://www.anaconda.com/download
Anti-Map2 Novus NB300-213 Immunofluorescence
Anti-mouse - Alexa 488 Thermo Fisher A11001 Immunofluorescence
Anti-rabbit - Alexa 555 Thermo Fisher A21429 Immunofluorescence
Anti-Tyrosine Hydroxylase Merck T2928 Immunofluorescence
Anti-α-synuclein Abcam 138501 Immunofluorescence
Bravo Automated Liquid Handling Platform with 384ST head Agilent If no liquid handler is available, the use of an electronic multichannel pipette is recommended.
Confocal microscope  Yokogawa CV7000 The use of an automated confocal fluorescence microscope is recommended to ensure image quality consistency.
Countess Automated cell counter Invitrogen Cell counting before seeding. Can also be done using a manual counting chamber.
DPBS +/+ Gibco 14040-133 Buffer for washing
EL406 Washer Dispenser  BioTek (Agilent)  If no liquid handler is available, the use of an electronic multichannel pipette is recommended.
Formaldehyde Solution (PFA 16 %) Euromedex EM-15710-S Fixation before staining
Hoechst 33342 Invitrogen H3570 Nuclear staining
iCell Base Medium 1 Fujifilm M1010 Base medium for neurons
iCell DPN, Donor#01279, Phenotype AHN, lot#106339, 1M Fujifilm C1087 Apparently healthy donor
iCell DPN, Donor#11299, Phenotype LRRK2 G2019S, phenotype PD lot#106139 Fujifilm C1149 Donor carrying LRRK2 G2019S mutation 
iCell Nervous System Supplement Fujifilm M1031 Supplement for base medium
iCell Neural Supplement B Fujifilm M1029 Supplement for base medium
Jupyter Python Notebook In-house development https://github.com/Ksilink/Notebooks/tree/main/Neuro/DopaNeuronProfiling Notebook to perform phenotypic profile visualization and classification from raw data.
Laminin Biolamina LN521 Plate coating
PFE-360 MedChemExpress HY-120085 LRRK2 kinase inhibitor
PhenoLink In-house development https://github.com/Ksilink/PhenoLink Software for image analysis
PhenoPlate 384w, PDL coated Perkin Elmer 6057500 Pre-coated plate for cell culture and imaging. This plate allows imaging of all wells using all objectives of the Yokogawa CV7000 microscope.
Storage plates Abgene 120 µL Thermo Scientific AB-0781 Necessary for compound dispensing using the Vprep pipetting system. If not available, the use of an electronic multichannel pipette is recommended.
Triton Sigma T9284 Permeabilization before lysis
Trypan Blue Sigma T8154-20ML Determination of living cells
Vprep Pipetting System  Agilent Medium change and compound dispensing. Alternatively, an electronic multichannel pipette can be used.

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