mouse cardiac progenitor cell isolation, digestion, and freezing

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 Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing at JoVE.com

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing  

Musens hjertestamcelleisolering, fordøjelse og frysning

Begynd med at placere embryonerne dissekeret fra den aflivede to til seks måneder gamle drægtige hunmus i et koldt komplet medium med 1% føtalt kvægserum. Efter fjernelse af endometrievæv, placenta og æggeblommesæk fra embryoet under 5X forstørrelse, dissekeres hjerteforfædrene fra det embryonale hjerteområde ved hjælp af tang. Træk alle hjerteområderne dissekeret fra fem museembryoner i et 1,5 ml mikrocentrifugerør og centrifuger dem i en svingende spand, forkølet til fire grader Celsius.Supernatanten fjernes, og vævet vaskes med 1 ml vævskulturklasse PBS. Centrifuger rørene ved 300G i et minut ved fire grader celsius. Derefter fjernes supernatanten, og to vaske gentages med en milliliter PBS.Når det er gjort, skal du udskifte PBS med 0,05% Trypsin-EDTA. Centrifuger og gentag to vaske med 0,05% Trypsin-EDTA. For at fordøje vævet tilsættes 50 mikroliter 0,05%Trypsin-EDTA og opvarmes i 10 minutter ved 37 grader Celsius.Påfør derefter en mild mekanisk dissociation efter fem minutter ved at opsuge suspensionen op og ned ved hjælp af en filterspids med bred åbning. Inaktiver Trypsin fordøjelsesaktivitet ved at tilføje 350 mikroliter komplet medium til de dissocierede celler. Før cellesuspensionen gennem en 40 mikrometer cellesi.Forbered cellerne til frysning ved centrifugering af den nyligt dissocierede cellesuspension. Supernatanten fjernes forsigtigt, og pelletsen resuspenderes i 1 ml kølet frysemedium. Overfør cellesuspensionen til et forkølet kryohætteglas, og anbring kryohætteglasset i en forkølet frysebeholder.Placer frysebeholderen ved minus 80 grader Celsius i fire til otte timer, før den overføres til flydende nitrogen til sekventeringsforsøg.

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Research

JoVE Journal

Developmental Biology

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing

343 Views.  Aix-Marseille University. Begin by placing the embryos dissected from the euthanized two to six-month-old pregnant female mouse in a cold complete medium with 1%fetal bovine serum. After removing the endometrial tissue, placenta, and yolk sack from the embryo under 5X magnification, dissect the cardiac progenitors from the embryonic heart region using forceps. Pull all the heart regions dissected from five mouse embryos in a 1.5 milliliter micro centrifuge tube and centrifuge them in a swinging bucket, pre-chilled to ...