thawing human ipsc-derived mda neurons for seeding

培养人中脑多巴胺能神经元进行表型分析

A variety of cell biological processes are disturbed in Parkinson&#39;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 profiling is an approach that involves creating a profile of a sample based on a collection of measurable characteristics, and second, it involves making predictions about a sample based on this profile2,3. The goal of profiling is to capture a diverse range of features, some of which may not have been previously associated with a disease or treatment3. As a result, profiling can reveal unexpected biological processes. Phenotypic profiling typically relies on fluorescently stained cells, and standardized assays, such as Cell Painting, have been developed to create phenotypic profiles4. Recently, phenotypic profiling has, for example, been applied for the characterization of small molecules or the accurate prediction of PD-subtypes solely based on patient-derived fibroblasts5,6. Despite these advances, phenotypic profiling has rarely been applied to post-mitotic differentiated cells, such as human induced pluripotent stem cell (iPSC)-derived mDA neurons that express PD-linked mutations such as LRRK2 G2019S. Significant challenges of iPSC-derived models include the presence of subtle or variable pathological features across differentiation batches or genotypes, and the fact that isolated PD phenotypes do not capture the full complexity of the disease. Furthermore, while iPSC neuronal models are physiologically relevant, they are rarely used in PD drug discovery processes due to concerns about technical complexity7,8.
We previously developed a robust methodology to measure multiple PD-related pathophysiological phenotypes in human mDA neurons that are both sensitive to genetic and chemical compound-induced phenotypic changes9. This article describes in detail a further optimized version of this methodology to create phenotypic profiles from mDA neurons (Figure 1). This protocol has several advantages over the previously described phenotypic profiling approaches, such as the use of high-quality mDA neurons and technical reproducibility. For the first time, this protocol enables phenotypic profiling in physiologically relevant post-mitotic mDA neurons after chemical perturbations in a highly scalable fashion. Fully differentiated and cryopreserved mDA neurons are commercially available, significantly decreasing batch-to-batch differentiation variability. Secondly, technical variability can be further reduced by using a well-defined experimental design (i.e., culture duration or avoiding edge wells), automated liquid handling and automated microscopy. Additionally, the initial steps of phenotypic profile analysis using unsupervised clustering or supervised classification approaches are outlined here, indicating how phenotypic profiling data can be analyzed. This protocol will be of use for researchers interested in phenotypic changes of mDA neurons induced by genetic or chemical perturbations, specifically when a highly scalable study setup is required, for example, during screening campaigns or when the effects of a smaller number of compounds are to be studied, for example, to determine toxic effects. In summary, it is anticipated that the application of phenotypic profiling of human neurons is a valuable technique to study complex disease-related phenotypes and characterize the cellular effects of drug candidates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65570/65570fig01.jpg" /> 
Figure 1: Schematic depiction of the experimental protocol to generate image-based phenotypic profiles from human iPSC-derived mDA neurons. <a href="https://www.jove.com/files/ftp_upload/65570/65570fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

作者聚焦：生成神经元表型谱 - 培养和成像人类中脑多巴胺能神经元的方案 

人类干细胞来源的中脑多巴胺能神经元的表型分析

We construct phenotypic profiles of differentiated human cells such as neurons. Our goal is to better understand the overall effects of chemical compound treatments on the cells. Phenotypic profiles can suggest less biased entry points for further mechanistic studies.The quality of protocols for differentiating human dopaminergic neurons has significantly improved, enabling the production of large batches of homogeneous neurons. Additionally, there is a rise in the use of lab automation for handling differentiated cells'reproducibility over extended periods, and of course, the exploitation of imaging-based data has become faster and more detailed. A big challenge is to ensure that phenotypic profiles are both quantifiable and reproducible.To achieve this, it is crucial to produce homogeneous neuron batches and identify an automation-suited protocol. However, with an automated protocols, there is always a trade-off between technical feasibility and physiological relevance. Here, we found a good trade-off.We developed a ready-to-use solution to thoroughly characterize human dopaminergic neurons using an imaging approach. The automated protocol, along with the image and data analysis workflow, will equip more scientists to create neuronal phenotypic profiles. We would like to better understand how phenotypic profiles differ between monogenetic and idiopathic forms of Parkinson's disease.We would also like to explore the changes in phenotypic aspects when dopaminergic neurons interact with other brain cell types such as microglia, for example.

Watch this Scientific Journal Video about Thawing Human iPSC-Derived mDA Neurons for Seeding at JoVE.com

解冻人 iPSC 衍生的 mDA 神经元进行接种

为了在播种当天解冻神经元，将水浴预热至37摄氏度。此外，将整个维护介质平衡到室温，同时保护其避光。同时，从液氮罐中取出含有商业获得的冷冻神经元的小瓶，并将其转移到干冰中。然后将小瓶放入 37 摄氏度的水浴中两分钟，以确保完全解冻。之后，使用70%乙醇对小瓶进行消毒。使用P1000移液器，将约370微升解冻的神经元从小瓶转移到50毫升离心管中。用 630 微升完全维持培养基冲洗空瓶。并使用移液器，将培养基以 45 度角滴入 50 毫升管中。以类似的方式，使用P1000移液器，向50毫升离心管中加入一毫升完全维持培养基。然后慢慢地将另外两毫升完全维持培养基加入管中。接下来，将 10 微升台盼蓝与 10 微升细胞悬液混合在微管中。然后将 10 微升混合物加入计数室载玻片中，用于计数细胞。细胞计数并将神经元转移到 15 毫升管中后，在室温下以 400g 离心 5 分钟。随后，除去上清液。使用P1000移液器，小心地将神经元沉淀重悬于一毫升完全维持培养基中。最后，加入必要体积的培养基以达到在同一天接种神经元所需的浓度。

thawing-human-ipsc-derived-mda-neurons-for-seeding

Research

JoVE Journal

Neuroscience

Thawing Human iPSC-Derived mDA Neurons for Seeding

167 次观看.  Ksilink. 为了在播种当天解冻神经元，将水浴预热至37摄氏度。此外，将整个维护介质平衡到室温，同时保护其避光。同时，从液氮罐中取出含有商业获得的冷冻神经元的小瓶，并将其转移到干冰中。然后将小瓶放入 37 摄氏度的水浴中两分钟，以确保完全解冻。之后，使用70%乙醇对小瓶进行消毒。使用P1000移液器，将约370微升解冻的神经元从小瓶转移到50毫升离心管中。用 6...

视频: 解冻人 iPSC 衍生的 mDA 神经元进行接种

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;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, &#945;-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.

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&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...

科研

神经科学

To thaw the neurons on the day of seeding, prewarm the water bath to 37 degrees Celsius. Also, equilibrate the complete maintenance media to room temperature while protecting it from light. Meanwhile, remove the vial containing the commercially-obtained frozen neurons from the liquid nitrogen tank and transfer it to dry ice.Then place the vial in the water bath at 37 degrees Celsius for two minutes to ensure complete thawing. Following which, disinfect the vial using 70%ethanol. Using a P1000 pipette, transfer the approximately 370 microliters of thawed neurons from the vial to a 50-milliliter centrifuge tube.Rinse the empty vial with 630 microliters of complete maintenance medium. And using a pipette, dispense the medium dropwise into the 50-milliliter tube at a 45-degree angle. In a similar manner, using a P1000 pipette, add one milliliter of complete maintenance medium to the 50-milliliter centrifuge tube.Then slowly add an additional two milliliters of complete maintenance medium into the tube. Next, mix 10 microliters of trypan blue with 10 microliters of the cell suspension in a micro tube. Then add 10 microliters of the mixture to a counting chamber slide for counting cells.Following cell counting and transferring the neurons to a 15-milliliter tube, centrifuge at 400g for five minutes at room temperature. Subsequently, remove the supernatant. Using a P1000 pipette, carefully resuspend the neuron pellet in one milliliter of complete maintenance medium.Finally, add the necessary volume of medium to achieve the desired concentration for seeding the neurons on the same day.

Human iPSC-Derived mDA Neurons Seeding and Compound Treatment for Phenotypic Profiling

To begin, take the Poly-D-Lysine pre-coated 384-well plate containing 25 microliters of laminin per well out of the refrigerator and equilibrate the plate to room temperature for about 30 minutes under the cell culture hood. Just before seeding, aspirate 15 microliters of coating solution from each well using an automated liquid handler or a 16-channel pipette. Then dispense 50 microliters of cell solution containing 300, 000 neurons per milliliter into each well.Avoid filling cells in columns 1, 2, 23, and 24, and rows A, B, O, and P to minimize possible edge effects. Fill those unused wells with 80 microliters of phosphate-buffered saline. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide.Prewarm an appropriate volume of complete maintenance medium at room temperature and away from light. Prepare a 1.5 times concentrated compound solution for all desired concentrations to be tested, using complete maintenance medium for dilutions. Using an automatic pipetting system, discard 40 microliters of medium per well from the neuron-containing plate, leaving 20 microliters of medium per well.Then add 40 microliters of the 1.5 times concentrated compound solution per well to achieve the desired final concentration. Following the desired protocol, healthy donor, or LRRK2 G2019S mutation-carrying midbrain dopaminergic neurons were successfully cultured for six days, and treated with either dimethyl sulfoxide or a LRRK2 kinase inhibitor. The neurons were stained with nuclear stain and antibodies against alpha-synuclein, tyrosine hydroxylase, and MAP2.

人 iPSC 衍生的 mDA 神经元接种和用于表型分析的复合处理

Image Processing of Fluorescently-Stained Human iPSC-Derived mDA Neurons for Phenotypic Profile Generation

To process the images of the fluorescently stained neurons acquired using a confocal fluorescence microscope use phenol link software for image segmentation and feature extraction. In the software load the plate of choice to access the data and select a desired image. To perform image segmentation on the illumination corrected raw images, adjust the fluorescent intensities to visualize the different channels.Empirically determine the respective channel intensity thresholds per plate to ensure the desired segmented signal corresponds to the signal in the raw image and minimize the background signal. To separate living from dead cells, define the nucleus size and intensity thresholds. Measure the size of the nucleus by double clicking and visualize the displayed intensity.When using 40 X images, maintain default parameters and run the processing. With the resulting tabular quantitative data construct phenotypic profiles to compare different cell lines or treatment conditions. Execute the Jupyter notebook cell by cell, using the Jupyter software for phenotypic profile generation and visualization.Images were segmented and phenotypic features were extracted. A total of 126 quantitative features that could be aggregated into well-based phenotypic profiles were determined. Some features showed changes upon compound treatment.For example, the alpha synuclein fluorescence intensity in tyrosine hydroxylase, or TH positive cells, decreased upon treatment with the LARK2 kinase inhibitor PFE-360. Other features, like the MAP2 neurite network length, or the ratio of TH positive neurons, presented differences only between the tested cell lines, but not upon compound treatment.