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

Kjerneisolasjon fra musehjertestamceller ved encellet oppløsning

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: Kjerneisolasjon fra musehjertestamceller for epigenom- og genekspresjonsprofilering ved encelleoppløsning  

Kjerneisolering fra musens hjerteprogenitorceller for epigenom og genuttrykksprofilering ved enkeltcelleoppløsning

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 hjertecelletining og fjerning av døde celler

Begynn med å plassere den frosne dissosierte embryonale hjertecellesuspensjonen som inneholder kryoflasker i et 37 grader Celsius vannbad i ett til to minutter. Tilsett en milliliter komplett medium, forvarmet ved 37 grader Celsius til tint suspensjon og bland den tre ganger med en pipette. Overfør deretter suspensjonen til et 15 milliliter konisk rør, som inneholder 10 millimeter varmt komplett medium.Før hele cellesuspensjonen over en 30 mikrometer sil og skyll silen med en til to milliliter varmt, komplett medium. Samle strømmen gjennom i samme koniske rør. Deretter sentrifugerer du rørene ved 300 G i fem minutter.Etter å ha fjernet supernatanten, vask pelleten med PBS og overfør cellesuspensjonen til et 1,5 milliliter mikrorør. Sentrifuger cellesuspensjonen og tilsett 100 mikroliter av de medfølgende magnetiske mikrokulene til de pelleterte cellene. Homogeniser suspensjonen fem ganger ved hjelp av en bred villsvinpipettespiss før 15 minutters inkubering ved romtemperatur.I mellomtiden skyller du den magnetiske separasjonskolonnen med 500 mikroliter bindingsbuffer. Når inkubasjonen er fullført, fortynn mikroperlecellesuspensjonsblandingen med 500 mikroliter bindingsbuffer. Deretter legger du til 0, 6 ml av cellesuspensjonen til den magnetiske separasjonskolonnen.Samle avløpsvannet fra levende celler i et 15 milliliter sentrifugerør. Skyll deretter 1,5 milliliter mikrorøret som inneholdt cellesuspensjonen ved hjelp av en milliliter bindingsbuffer. Etter skylling, overfør bindingsbufferen fra 1,5 milliliter mikrorør til kolonnen og samle avløpet i samme 15 milliliter rør.Gjenta skyllingen av 1,5 milliliter røret med en milliliter bindingsbuffer. Etter sentrifugering av avløpsvannet ved 300 G i fem minutter, fjern supernatanten uten å forstyrre cellepelleten. Tilsett en milliliter PBS BSA-oppløsningen og bland forsiktig ved pipettering med en bred åpningspipespiss.Overfør deretter cellesuspensjonen til et 1,5 milliliter mikrorør. Igjen, sentrifuger cellesuspensjonen og fjern supernatanten uten å forstyrre cellepelleten. Gjenta de to vaskene på en milliliter PBS BSA.Etter å ha fjernet en milliliter PBS BSA, resuspendere cellene i 100 mikroliter PBS BSA-løsning. Bland det ved å pipettere 10 ganger. Bestem konsentrasjonen ved å utføre en trypanblå analyse for å sikre et celletall på mer enn eller lik 100 000 celler og utfør en fluorescensbasert vurdering for cellens levedyktighet.Det ekstra trinnet med sortering og fjerning av de døde cellene etter tining tillot anskaffelse av en renere cellesuspensjon med betydelig høyere cellelevedyktighet og forbedret kvaliteten på startprøven. For mer nøyaktighet ble to forskjellige tellemetoder brukt til å kvantifisere cellene og kjernene, inkludert trypanblå og fluorescensbasert vurdering av levedyktigheten. I denne protokollen ble det observert at cellens levedyktighet gikk fra 80 til 90% før kjerneisolering til mindre enn 5% etter kjerneisolering.

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 ...