mouse cardiac progenitor cell isolation, digestion, and freezing

Aislamiento de núcleos de células progenitoras cardíacas de ratón con resolución de una sola célula

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.

Autor destacado: Aislamiento de núcleos de células progenitoras cardíacas de ratón para el perfil de epigenoma y expresión génica a resolución de una sola célula  

Aislamiento de núcleos de células progenitoras cardíacas de ratón para epigenoma y perfil de expresión génica a resolución de una sola célula

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  

Aislamiento, digestión y congelación de células progenitoras cardíacas de ratón

Comience colocando los embriones disecados de la ratona hembra embarazada sacrificada de dos a seis meses de edad en un medio completo frío con suero bovino fetal al 1%. Después de extraer el tejido endometrial, la placenta y el saco vitelino del embrión con un aumento de 5X, disecciona los progenitores cardíacos de la región embrionaria del corazón con fórceps. Extraiga todas las regiones del corazón diseccionadas de cinco embriones de ratón en un tubo de microcentrífuga de 1,5 mililitros y centrifugarlos en un cubo oscilante, previamente enfriado a cuatro grados centígrados.Retire el sobrenadante y lave el tejido con un mililitro de PBS de grado de cultivo de tejido. Centrifugar los tubos a 300G durante un minuto a cuatro grados centígrados. Luego, retire el sobrenadante y repita dos lavados con un mililitro de PBS.Una vez hecho esto, reemplace el PBS con 0.05% Tripsina-EDTA. Centrifugar y repetir dos lavados con 0,05% de tripsina-EDTA. Para digerir el tejido, agregue 50 microlitros de 0.05% de tripsina-EDTA y caliente durante 10 minutos a 37 grados centígrados.A continuación, aplique una disociación mecánica suave después de cinco minutos aspirando la suspensión hacia arriba y hacia abajo con una punta de filtro de orificio ancho. Inactivar la actividad digestiva de tripsina añadiendo 350 microlitros de medio completo a las células disociadas. Pase la suspensión celular a través de un filtro celular de 40 micrómetros.Prepare las células para la congelación centrifugando la suspensión celular recién disociada. Retire con cuidado el sobrenadante y vuelva a suspender el pellet en un mililitro de medio de congelación refrigerado. Transfiera la suspensión celular a un vial criogénico preenfriado y coloque el vial criogénico en un recipiente de congelación preenfriado.Coloque el recipiente de congelación a menos 80 grados centígrados durante cuatro a ocho horas antes de transferirlo a nitrógeno líquido para experimentos de secuenciación.

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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 ...