embryonic heart cell thawing and removal of dead cells

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise to different subtypes of cardiac cells, including myocardial, fibroblast, epicardial, and endocardial cells4,5.&#160;Single-cell genomics is emerging as a key method for studying heart development and assessing the impact of cellular heterogeneity in health and disease6. The development of multi-omics methods for the simultaneous measurement of different parameters and the expansion of computational pipelines has facilitated the discovery of cell types and subtypes in the normal and diseased heart6. This article describes a reliable single-nucleus isolation protocol for frozen cardiac progenitor cells obtained from mouse embryos that is compatible with downstream snRNA-seq and snATAC-seq (as well as snRNA-seq and snATAC-seq combined)7,8,9.
ATAC-seq is a robust method that allows the identification of regulatory open chromatin regions and the positioning of nucleosomes10,11. This information is used to draw conclusions about the location, identity, and activity of transcription factors. The activity of chromatin factors, including remodelers, as well as the transcriptional activity of RNA polymerase, can, thus, be analyzed since the method is highly sensitive for measuring quantitative changes in chromatin structure1,2. Thus, ATAC-seq provides a robust and impartial approach to uncovering the mechanisms controlling transcriptional regulation in a specific cell type. ATAC-seq protocols have also been validated to measure chromatin accessibility in single cells, revealing variability in the chromatin architecture within cell populations10,12,13.
Although there have been notable advances in the field of single cells in recent years, the main difficulty is the processing of the fresh samples needed to perform these experiments14. To circumvent this difficulty, various tests have been carried out with the aim of conducting analyses such as snRNA-seq and snATAC-seq with frozen cardiac tissue or cells15,16.
Several platforms have been used to analyze single-cell genomics data17. The widely used platforms for single-cell gene expression and ATAC profiling are platforms for multiple microfluidic droplet encapsulation17. As these platforms use microfluidic chambers, debris or aggregates can clog the system, resulting in non-usable data. Thus, the success of single-cell studies depends on the accurate isolation of individual cells/nuclei.
The protocol presented here uses a similar approach to recent studies using snRNA-seq and snATAC-seq to understand congenital heart defects18,19,20,21,22,23. This procedure utilizes the enzymatic dissociation of freshly microdissected cardiac tissue followed by the cryopreservation of mouse cardiac progenitor cells. After thawing, the viable cells are purified and processed for nuclear isolation. In this work, this protocol was successfully used to obtain snRNA-seq and snATAC-seq data from the same nuclear preparation of mouse cardiac progenitor cells.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches such as single nuclei RNA-seq and single nuclei ataxic are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been a significant progress in the field in single cell for recent years, the main difficulty is processing free samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defects.This protocol describes the steps to follow for successfully isolating high quality nuclei from frozen cardiac cell suspension obtained from mouse embryos. And our protocol is compatible with downstream single nuclei RNA-seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research.

Watch this Scientific Journal Video about Embryonic Heart Cell Thawing and Removal of Dead Cells at JoVE.com

Embryonic Heart Cell Thawing and Removal of Dead Cells  

Begin by placing the frozen dissociated embryonic heart cell suspension containing cryo vials in a 37 degrees Celsius water bath for one to two minutes. Add one milliliter of complete medium, pre warmed at 37 degrees Celsius to the thawed suspension and mix it three times with a pipette. Then transfer the suspension to a 15 milliliter conical tube, containing 10 millimeters of warm complete medium.Pass the entire cell suspension over a 30 micrometer strainer and rinse the strainer with one to two milliliters of warm, complete medium. Collect the flow through in the same conical tube. Next, centrifuge the tubes at 300 G for five minutes.After removing the supernatant, wash the pellet with PBS and transfer the cell suspension into a 1.5 milliliter micro tube. Centrifuge the cell suspension and add 100 microliters of the provided magnetic microbeads to the pelleted cells. Homogenize the suspension five times using a wide boar pipette tip before 15 minutes of incubation at room temperature.In the meantime, rinse the magnetic separation column with 500 microliters of binding buffer. Once the incubation is complete, dilute the microbead cell suspension mixture using 500 microliters of binding buffer. Next, add 0.6 milliliters of the cell suspension to the magnetic separation column.Collect the live cell effluent in a 15 milliliter centrifuge tube. Next, rinse the 1.5 milliliter micro tube that contained the cell suspension using one milliliter of binding buffer. After rinsing, transfer the binding buffer from the 1.5 milliliter micro tube to the column and collect the effluent in the same 15 milliliter tube.Repeat the rinsing of the 1.5 milliliter tube using one milliliter of binding buffer. After centrifuging the effluent at 300 G for five minutes, remove the supernatant without disrupting the cell pellet. Add one milliliter of the PBS BSA solution and gently mix by pipetting using a wide orifice pipette tip.Then transfer the cell suspension into a 1.5 milliliter micro tube. Again, centrifuge the cell suspension and remove the supernatant without disrupting the cell pellet. Repeat the two washes of one milliliter of PBS BSA.After removing the one milliliter of PBS BSA, resuspend the cells in 100 microliters of PBS BSA solution. Mix it by pipetting 10 times. Determine the concentration by performing a trypan blue assay to ensure a cell count of more than or equal to 100, 000 cells and perform a fluorescence based assessment for the viability of the cells.The additional step of sorting and removing the dead cells after thawing allowed the procurement of a cleaner cell suspension with significantly higher cell viability and improved the quality of the starting sample. For more accuracy, two different counting methods were used to quantify the cells and nuclei, including trypan blue and the fluorescence based assessment of the viability. In this protocol, it was observed that the cell viability passed from 80 to 90%before nuclei isolation to less than 5%after nuclei isolation.

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Research

JoVE Journal

Developmental Biology

357 Görüntüleme.  Aix-Marseille University. Kriyo şişeleri içeren dondurulmuş ayrışmış embriyonik kalp hücresi süspansiyonunu bir ila iki dakika boyunca 37 santigrat derecelik bir su banyosuna yerleştirerek başlayın. Çözülmüş süspansiyona 37 santigrat derecede önceden ısıtılmış bir mililitre tam ortam ekleyin ve bir pipetle üç kez karıştırın. Daha sonra süspansiyonu, 10 milimetre sıcak tam ortam içeren 15 mililitrelik bir konik tüpe aktarın.Tüm hücre süspansiyonunu 30 mikrometrelik bir süz...