Name: Embryonic Heart Cell Thawing and Removal of Dead Cells
Uploaded: 2023-05-12T14:30:00
Description: 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

Kerneisolering fra musehjertestamceller ved enkeltcellet opløsning

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.

Forfatter Spotlight: Kerneisolering fra musehjertestamceller til epigenom- og genekspressionsprofilering ved enkeltcelleopløsning  

Kerneisolering fra musehjertestamceller til epigenom og genekspressionsprofilering ved enkeltcelleopløsning

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  

Embryonal optøning af hjerteceller og fjernelse af døde celler

Begynd med at placere den frosne dissocierede embryonale hjertecellesuspension indeholdende kryohætteglas i et 37 grader Celsius vandbad i et til to minutter. Tilsæt en milliliter komplet medium, forvarmet til 37 grader Celsius til den optøede suspension og bland den tre gange med en pipette. Overfør derefter suspensionen til et 15 ml konisk rør, der indeholder 10 millimeter varmt komplet medium.Før hele cellesuspensionen over en 30 mikrometer sil og skyl silen med en til to ml varm, komplet medium. Saml strømmen igennem i det samme koniske rør. Derefter centrifugeres rørene ved 300 G i fem minutter.Efter fjernelse af supernatanten vaskes pelleten med PBS og cellesuspensionen overføres til et 1,5 ml mikroglas. Centrifuger cellesuspensionen og tilsæt 100 mikroliter af de medfølgende magnetiske mikroperler til de pelleterede celler. Suspensionen homogeniseres fem gange ved hjælp af en bred ornepipettespids inden 15 minutters inkubation ved stuetemperatur.I mellemtiden skylles den magnetiske separationskolonne med 500 mikroliter bindingsbuffer. Når inkubationen er afsluttet, fortyndes mikroperlecellesuspensionsblandingen ved hjælp af 500 mikroliter bindingsbuffer. Derefter tilsættes 0,6 ml af cellesuspensionen til den magnetiske separationskolonne.Det levende celleudløb opsamles i et 15 ml centrifugerør. Skyl derefter det 1,5 ml mikrorør, der indeholdt cellesuspensionen, ved hjælp af en milliliter bindingsbuffer. Efter skylning overføres bindingsbufferen fra 1,5 ml mikrorøret til søjlen og opsamles i det samme 15 ml rør.Gentag skylningen af 1,5 ml røret ved hjælp af en milliliter bindingsbuffer. Efter centrifugering af spildevandet ved 300 G i fem minutter fjernes supernatanten uden at forstyrre cellepellet. Der tilsættes en milliliter PBS BSA-opløsning, og blandingen blandes forsigtigt ved pipettering med en pipettespids med bred åbning.Overfør derefter cellesuspensionen til et 1,5 ml mikrorør. Igen centrifugeres cellesuspensionen, og supernatanten fjernes uden at forstyrre cellepellet. Gentag de to vaske på en milliliter PBS BSA.Efter fjernelse af den ene milliliter PBS BSA resuspenderes cellerne i 100 mikroliter PBS BSA-opløsning. Bland det ved pipettering 10 gange. Bestem koncentrationen ved at udføre et trypanblåt assay for at sikre et celletal på mere end eller lig med 100.000 celler og udfør en fluorescensbaseret vurdering af cellernes levedygtighed.Det ekstra trin med sortering og fjernelse af de døde celler efter optøning gjorde det muligt at udtage en renere cellesuspension med signifikant højere cellelevedygtighed og forbedrede kvaliteten af startprøven. For mere nøjagtighed blev to forskellige tællemetoder brugt til at kvantificere cellerne og kernerne, herunder trypanblåt og den fluorescensbaserede vurdering af levedygtigheden. I denne protokol blev det observeret, at cellelevedygtigheden gik fra 80 til 90% før kerneisolering til mindre end 5% efter kerneisolering.

Research

JoVE Journal

Developmental Biology

Embryonic Heart Cell Thawing and Removal of Dead Cells

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

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...