Cells of the adult human heart methods
Aim. Evidence-backed execution summary for Cells of the adult human heart methods from Cells of the adult human heart.
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human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Cardiomyocyte heterogeneity
reagent used in the protocol.
- Use
- Ventricular cardiomyocytes are enriched in genes encoding sarcomere proteins ( MYH7 and MYL2 ), transcription factors ( IRX3, IRX5, IRX6, MASP1 and HEY2 ), and PRDM16, mutated in left ventricular non-compaction cardiomyopathy. Other abundant transcripts enable tissue integrity despite high ventricular strain: P...
Conduction system and neuronal cells
reagent used in the protocol.
- Use
- Among 3,961 cells expressing prototypic electrophysiologic transcripts ( NRXN1, NRXN3 and KCNMB4 ), we identify six neuronal cell subclusters (Extended Data Fig. ). NC1 constitutes 75-80% of neuronal cells and exhibits a basal gene program including LGI4, required for glia development and axon myelination....
Single nuclei isolation
reagent used in the protocol.
- Use
- Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM suc...
Single-cell preparation
reagent used in the protocol.
- Use
- Heart tissues (0.2-0.9 g) were transferred from cardioplegic solution into gentleMACS C-tubes (Miltenyi Biotec) containing enzymatic digestion base solution (100 µg ml -1 liberase TH Research grade and 50 µg ml -1 DNase I, HBSS 10 mM HEPES and 30 mM taurine). Tissues were minced using sc...
CD45 + cell enrichment
reagent used in the protocol.
- Use
- Cell suspension was prepared as described above and subsequently labelled using anti-human CD45 monoclonal antibody-conjugated microbeads according to the manufacturer's protocol (Miltenyi Biotec). In brief, up to 10 7 cells were incubated for 15 min at 4 °C in 80 µl of PBS, BSA, EDTA buffer (1...
Chromium 10X library preparation
reagent used in the protocol.
- Use
- Single cells and nuclei were manually counted by Trypan blue exclusion or automatically using a Countess II (Life Technologies) using at least two separate counts. Cell or nuclei suspension was adjusted to 400-1,000 cells per microlitre and loaded on the Chromium Controller (10X Genomics) with a targeted cell...
Spatial validation using smFISH with RNAscope probes
reagent used in the protocol.
- Use
- During preparation of formalin-fixed paraffin-embedded (FFPE) samples fresh tissue was fixed in neutral-buffered 10% formalin for 18-36 h and subsequently embedded in paraffin blocks. Fixed-frozen tissue samples were fixed in 4% paraformaldehyde (ThermoFisher). Sections were cut at 5-µm thickness using a...
Acquisition of skeletal muscle tissue
reagent used in the protocol.
- Use
- Intercostal muscle samples were obtained from between the second and third rib on the left side. This is typically from the deepest layer of muscle (furthest away from the skin). Samples were collected directly into the cold preservation solution.
Main
The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is propelled into the lungs. Oxygenated blood enters the left atrium and ventricle, which propels blood across the body at systemic pressure....
- Use
- The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is propelled into the lungs. Oxygenated blood enters the left atrium and ventricle, which propels blood across the body at systemic pressure....
Main
Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq, respectively) and multiplex single-molecule fluorescence in situ hybridization (smFISH) enable the identification of anatomical specificities, molecular signatures, intercellular networks and spatial relationships by illuminating the coordinated...
- Use
- Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq, respectively) and multiplex single-molecule fluorescence in situ hybridization (smFISH) enable the identification of anatomical specificities, molecular signatures, intercellular networks and spatial relationships by illuminating the coordinated...
Cardiomyocyte heterogeneity
Cardiomyocytes show high-level expression of genes that encode contractile force-generating sarcomere proteins ( TTN, MYBPC3 and TNNT2 ) and calcium-mediated processes ( RYR2, PLN and SLC8A1 ). Consistent with bulk tissue RNA-sequencing (RNA-seq) data, we observe markedly distinct transcriptional signatures in ve...
- Use
- Cardiomyocytes show high-level expression of genes that encode contractile force-generating sarcomere proteins ( TTN, MYBPC3 and TNNT2 ) and calcium-mediated processes ( RYR2, PLN and SLC8A1 ). Consistent with bulk tissue RNA-sequencing (RNA-seq) data, we observe markedly distinct transcriptional signatures in ve...
Cardiomyocyte heterogeneity
vCM5 (approximately 1%) comprises cells with high levels of DLC1 and EBF2, regulating brown adipocyte differentiation and perhaps cardiac pacemaker activity, and transcripts identified in neuronal lineages ( SOX5, EBF1 and KCNAB1 ). As EBF1- depleted mice have a profoundly hypoplastic ventricular conduction system...
- Use
- vCM5 (approximately 1%) comprises cells with high levels of DLC1 and EBF2, regulating brown adipocyte differentiation and perhaps cardiac pacemaker activity, and transcripts identified in neuronal lineages ( SOX5, EBF1 and KCNAB1 ). As EBF1- depleted mice have a profoundly hypoplastic ventricular conduction system...
Cardiac fibroblasts
Cells of the FB compartment show enriched expression of DCN, GSN and PDGFRA within seven populations (Fig. ) with regional enrichment in ventricles (FB1) and atria (FB2). This is consistent with distinctive functional properties, including stronger profibrotic responses, by atrial FBs. FB1 and FB2 cells express ca...
- Use
- Cells of the FB compartment show enriched expression of DCN, GSN and PDGFRA within seven populations (Fig. ) with regional enrichment in ventricles (FB1) and atria (FB2). This is consistent with distinctive functional properties, including stronger profibrotic responses, by atrial FBs. FB1 and FB2 cells express ca...
Cardiac fibroblasts
Separate clustering of atrial and ventricular FBs recapitulated the populations described above, including an OSM-signalling population in each chamber (aFB4 and vFB3). In addition, we identify distinct chamber-specific ECM-producing FBs that differ in the expression of collagen isoforms and other ECM-related (aFB2...
- Use
- Separate clustering of atrial and ventricular FBs recapitulated the populations described above, including an OSM-signalling population in each chamber (aFB4 and vFB3). In addition, we identify distinct chamber-specific ECM-producing FBs that differ in the expression of collagen isoforms and other ECM-related (aFB2...
Conduction system and neuronal cells
Among 3,961 cells expressing prototypic electrophysiologic transcripts ( NRXN1, NRXN3 and KCNMB4 ), we identify six neuronal cell subclusters (Extended Data Fig. ). NC1 constitutes 75-80% of neuronal cells and exhibits a basal gene program including LGI4, required for glia development and axon myelination....
- Use
- Among 3,961 cells expressing prototypic electrophysiologic transcripts ( NRXN1, NRXN3 and KCNMB4 ), we identify six neuronal cell subclusters (Extended Data Fig. ). NC1 constitutes 75-80% of neuronal cells and exhibits a basal gene program including LGI4, required for glia development and axon myelination....
Single nuclei isolation
Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM suc...
- Use
- Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM suc...
Gene Ontology enrichment analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For the ventricular cardiomyocyte population, we used the R package gProfileR ( https://cran.r-project.org/web/packages/gProfileR/index.html ) with the score-ranked gene list of vCM4 as input and the set of genes expressed in ventricular cardiomyocytes as background (those genes having a UMI count >1). To perform th...
Statistics and reproducibility
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All analyses were performed using R Software, v.3.6.1. Student's t -tests were used to compare cell type distributions at each site. P < 0.05 was considered statistically significant. Linear regression models (correlations) were obtained using the R linear model function (lm), which estimates statis...
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COVID-19 and GWAS disease relevance
We define cells enriched for genes from 12 cardiovascular genome-wide association studies (GWAS) and involved in SARS-CoV-2 infection using MAGMA (Extended Data Fig. ). Atrial fibrillation GWAS signals are associated with transcriptional profiles in vCM3, owing to higher mean expression of CAV1, CAV2 and PRRX1. PR interval GWAS signals are associated with vCM3 and aCM5, with high expression of SCN5A, CAV1, ARHGAP24, MEIS1, TBX5 and TTN. GWAS signals for QRS duration are associated with specific gene expression in NC2 ( PRKCA, CEP85L, SLC35F1, SIPA1L1, KLF12 and FADS2 ). Coronary artery disease and hypertension GWAS signals are associated with transcripts from many cell lineages, particularly SMCs, FBs, and ECs, reflecting the relevance of vascular cells in both disorders.
Research ethics for donor tissues
Heart tissues (donors D1-D7 and D11) were processed at Wellcome Sanger Institute (Hinxton, UK) and obtained from deceased transplant organ donors after Research Ethics Committee approval (ref 15/EE/0152, East of England Cambridge South Research Ethics Committee) and informed consent from the donor families. Heart tissues (donors H2-H7) were processed at Harvard Medical School (Boston, Massachusetts, USA) and obtained from deceased organ donors after Human Research Ethics Board approval Pro00011739 (University of Alberta, Edmonton, Canada). Informed consent from donor families was acquired via the institutional Human Organ Procurement and Exchange Program (HOPE). Cardiovascular history was unremarkable for all donors (Supplementary Table ).
Single nuclei isolation
Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl 2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1 × protease inhibitor, 0.4 U µl -1 RNaseIn, 0.2 U µl -1 SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40-µm cell strainer (Corning). After centrifugation (500 g, 5 min, 4 °C) the supernatant was removed and the pellet was resuspended in storage buffer (1 × PBS, 4% bovine serum albumin (BSA), 0.2 U µl -1 Protector RNaseIn). Nuclei were stained with NucBlue Live ReadyProbes Reagents (ThermoFis...
Single-cell preparation
Heart tissues (0.2-0.9 g) were transferred from cardioplegic solution into gentleMACS C-tubes (Miltenyi Biotec) containing enzymatic digestion base solution (100 µg ml -1 liberase TH Research grade and 50 µg ml -1 DNase I, HBSS 10 mM HEPES and 30 mM taurine). Tissues were minced using scissors (FST) and automatically digested using gentleMACS Octo Dissociator (Miltenyi Biotec) with heaters. Cardiomyocyte-depleted single-cell suspension were washed with base solution containing 20% fetal bovine serum (FBS) (Gibco), filtered through 70-µm nylon strainer (BD Falcon), collected by centrifugation (330 g, 10 min, 4 °C) and resuspended in base solution containing 0.2% FBS (Gibco). Cells were manually counted three times by Trypan blue exclusion after each centrifugation and resuspended at a concentration of at least 2 × 1...
CD45 + cell enrichment
Cell suspension was prepared as described above and subsequently labelled using anti-human CD45 monoclonal antibody-conjugated microbeads according to the manufacturer's protocol (Miltenyi Biotec). In brief, up to 10 7 cells were incubated for 15 min at 4 °C in 80 µl of PBS, BSA, EDTA buffer (1 × PBS pH 7.2, 0.5% BSA, 2 mM EDTA) containing 20 µl CD45 microbeads. Cell suspension was washed in PBS, BSA, EDTA buffer once and collected by centrifugation (330 g, 10 min, 4 °C). Resuspended cells were applied to MACS LS columns (Miltenyi Biotec). CD45-depleted cell fraction was discarded after three washes with PBS, BSA and EDTA buffer and the CD45 + cell fraction was collected in PBS, BSA and EDTA buffer by removal of the columns from the magnetic field. CD45 + cells were counted and resuspended in PBS, BSA and EDTA buffer to a concentration of...
Nuclei isolation for skeletal muscle
Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and minced to obtain fragments of approximately 1 mm 3. Per sample, approximately 0.3 g of minced tissues was homogenized in 3 ml of buffer A (250 mM sucrose, 10 mg ml -1 BSA, 5 mM MgCl 2, 0.12 U µl -1 RNaseIn, 0.06 U µl -1 SUPERasIn, 1 × protease inhibitor) using Dounce tissue grinder set (Merck) with 50 strokes of the loose pestle (A). The homogenate was filtered through a 100-µm cell strainer (Corning) and the strainer was washed with 1 ml and then 750 µl of buffer A. After the addition of Triton X-100 (final concentration 0.5%), the mixture was further homogenized with 50 strokes of the tight pestle (B). After filtering through a 40-µm strainer, nuclei were centrifuged (3,000 g, 5 min, 4 °C), resuspended in 1 ml of buffer B (320 mM sucro...
Single-cell isolation for skeletal muscle
Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and finely minced. Then, 2 g of the minced tissue was transferred to digestion buffer 1 (750 U ml -1 collagenase type 2 in 1 × PBS) and incubated at 37 °C in a water bath for 90 min. The partially digested tissue was collected by centrifugation (650 g, 5 min, 4 °C) and the pellet was resuspended in digestion buffer 2 (100 U ml -1 collagenase type 2, 2 U ml -1 dispase in PBS). After 30 min incubation at 37 °C in a water bath, the digestion was stopped by the addition of 2% FBS. Cells were filtered through a 100-µm and a 40-µm nylon strainer (BD Falcon), collected by centrifugation (650 g, 4 °C, 3 min) and washed with 1 × PBS, 2% FBS. Subsequently, a 20% Percoll gradient (15,000 g, 4 °C, 20 min) was used for ce...
Extended data figures and tables
Extended Data Fig. 10 Relevance for COVID-19 and GWAS studies. a, Global expression (log 2 FC) of ACE2 in all cardiac cells. b - d, Gene expression of ACE2, TMPRSS2, CTSB and CTSL in cardiomyocytes ( b ), FBs ( c ) and vascular cells ( d ). e, Multiplexed smFISH expression of DCN (cyan), KCNJ8 (green) and ACE2 (red), nuclei are DAPI-stained (dark blue) marking fibroblasts (#; expression of DCN ) and pericytes (*; co-expression of DCN and KCNJ8 ) in right ventricular tissue section. Scale bars, 5 µm. For statistics and reproducibility, see. f, The colour coding of the heat map shows the -log 10 ( P value) of the MAGMA GWAS enrichment analysis for the association between cell type-specific expression ( y axis) and GWAS signals ( x axis). The cell types refer to the subcluster annotations and GWAS studies refer to Supplementary Table. AF, atrial fibri...
Measurement outputs
What raw and processed outputs should exist?
After removal of empty droplets, we applied scrublet to assign a doublet score (scrublet_score) to each cell. These cells were clustered and visualized using the UMAP method. I...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The heart is derived from multipotent progenitor cells that comprise two heart fields. Cells of the first heart field primarily populate the left ventricle; second heart field c...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
ACM3 and vCM3 share similar transcriptional profiles including enrichment of the SMC gene CNN1 (verified by smFISH) (Fig., Extended Data Fig. ). The molecular signatures of aCM...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig.
from paperScoring or quantification
Quantify the primary readouts for this experiment: After removal of empty droplets, we applied scrublet to assign a doublet score (scrublet_score) to each cell. These cells were clustered and visualized using the UMAP method. I...; The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is...; The heart is derived from multipotent progenitor cells that comprise two heart fields. Cells of the first heart field primarily populate the left ventricle; second heart field c...; ACM3 and vCM3 share similar transcriptional profiles including enrichment of the SMC gene CNN1 (verified by smFISH) (Fig., Extended Data Fig. ). The molecular signatures of aCM....
from paperStatistical comparison
The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is...; We identify five ventricular cardiomyocyte (vCM1-vCM5) populations: vCM1 comprise 63.9% of left ventricular cardiomyocytes but only 36.7% of right ventricular cardiomyocyt...; The vascular compartment includes 17 distinct populations of EC, SMC, pericyte, mesothelial cells with anatomical and arterio-venous specificities (Fig., Supplementary Tables,...; No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcom...
from paperReporting output
Report representative outputs alongside summary comparisons for After removal of empty droplets, we applied scrublet to assign a doublet score (scrublet_score) to each cell. These cells were clustered and visualized using the UMAP method. I..., The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is..., The heart is derived from multipotent progenitor cells that comprise two heart fields. Cells of the first heart field primarily populate the left ventricle; second heart field c..., ACM3 and vCM3 share similar transcriptional profiles including enrichment of the SMC gene CNN1 (verified by smFISH) (Fig., Extended Data Fig. ). The molecular signatures of aCM....
inferred from protocolStructured statistical methods
The heart is a complex organ, composed of four morphologically and functionally distinct chambers (Fig. ). Deoxygenated blood from the low-pressure right atrium and ventricle is...; We identify five ventricular cardiomyocyte (vCM1-vCM5) populations: vCM1 comprise 63.9% of left ventricular cardiomyocytes but only 36.7% of right ventricular cardiomyocyt...; The vascular compartment includes 17 distinct populations of EC, SMC, pericyte, mesothelial cells with anatomical and arterio-venous specificities (Fig., Supplementary Tables,...; No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcom...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
We define cells enriched for genes from 12 cardiovascular genome-wide association studies (GWAS) and involved in SARS-CoV-2 infection using MAGMA (Extended Data Fig. ). Atrial fibrillation GWAS signals are associated with transcriptional profiles in vCM3, owing to higher mean expression of CAV1, CAV2 and PRRX1. PR interval GWAS signals are associated with vCM3 and aCM5, with high expression of SCN5A, CAV1, ARHGAP24, MEIS1, TBX5 and TTN. GWAS signals for QRS duration are associated with specific gene expression in NC2 ( PRKCA, CEP85L, SLC35F1, SIPA1L1, KLF12 and FADS2 ). Coronary artery disease and hypertension GWAS signals are associated with transcripts from many cell lineages, particularly SMCs, FBs, and ECs, reflecting the relevance of vascular cells in both disorders.
Heart tissues (donors D1-D7 and D11) were processed at Wellcome Sanger Institute (Hinxton, UK) and obtained from deceased transplant organ donors after Research Ethics Committee approval (ref 15/EE/0152, East of England Cambridge South Research Ethics Committee) and informed consent from the donor families. Heart tissues (donors H2-H7) were processed at Harvard Medical School (Boston, Massachusetts, USA) and obtained from deceased organ donors after Human Research Ethics Board approval Pro00011739 (University of Alberta, Edmonton, Canada). Informed consent from donor families was acquired via the institutional Human Organ Procurement and Exchange Program (HOPE). Cardiovascular history was unremarkable for all donors (Supplementary Table ).
Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl 2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1 × protease inhibitor, 0.4 U µl -1 RNaseIn, 0.2 U µl -1 SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40-µm cell strainer (Corning). After centrifugation (500 g, 5 min, 4 °C) the supernatant was removed and the pellet was resuspended in storage buffer (1 × PBS, 4% bovine serum albumin (BSA), 0.2 U µl -1 Protector RNaseIn). Nuclei were stained with NucBlue Live ReadyProbes Reagents (ThermoFisher) and Hoechst-positive single nuclei were purified by fluorescent activated cell sorting (FACS) using influx, XDP or FACSAria (BD Biosciences) (Supplementary Fig. ). Nuclei purification and integrity was verified under a microscope, and nuclei were further processed using the Chromium Controller...
Heart tissues (0.2-0.9 g) were transferred from cardioplegic solution into gentleMACS C-tubes (Miltenyi Biotec) containing enzymatic digestion base solution (100 µg ml -1 liberase TH Research grade and 50 µg ml -1 DNase I, HBSS 10 mM HEPES and 30 mM taurine). Tissues were minced using scissors (FST) and automatically digested using gentleMACS Octo Dissociator (Miltenyi Biotec) with heaters. Cardiomyocyte-depleted single-cell suspension were washed with base solution containing 20% fetal bovine serum (FBS) (Gibco), filtered through 70-µm nylon strainer (BD Falcon), collected by centrifugation (330 g, 10 min, 4 °C) and resuspended in base solution containing 0.2% FBS (Gibco). Cells were manually counted three times by Trypan blue exclusion after each centrifugation and resuspended at a concentration of at least 2 × 10 6 ml -1. Single cells were processed using Chromium Controller (10X Genomics) according to the manufacturer's protocol.
Cell suspension was prepared as described above and subsequently labelled using anti-human CD45 monoclonal antibody-conjugated microbeads according to the manufacturer's protocol (Miltenyi Biotec). In brief, up to 10 7 cells were incubated for 15 min at 4 °C in 80 µl of PBS, BSA, EDTA buffer (1 × PBS pH 7.2, 0.5% BSA, 2 mM EDTA) containing 20 µl CD45 microbeads. Cell suspension was washed in PBS, BSA, EDTA buffer once and collected by centrifugation (330 g, 10 min, 4 °C). Resuspended cells were applied to MACS LS columns (Miltenyi Biotec). CD45-depleted cell fraction was discarded after three washes with PBS, BSA and EDTA buffer and the CD45 + cell fraction was collected in PBS, BSA and EDTA buffer by removal of the columns from the magnetic field. CD45 + cells were counted and resuspended in PBS, BSA and EDTA buffer to a concentration of at least 2 × 10 6 per ml before further processing using a Chromium Controller (10X Genomics) according to the manufacturer's protocol.
Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and minced to obtain fragments of approximately 1 mm 3. Per sample, approximately 0.3 g of minced tissues was homogenized in 3 ml of buffer A (250 mM sucrose, 10 mg ml -1 BSA, 5 mM MgCl 2, 0.12 U µl -1 RNaseIn, 0.06 U µl -1 SUPERasIn, 1 × protease inhibitor) using Dounce tissue grinder set (Merck) with 50 strokes of the loose pestle (A). The homogenate was filtered through a 100-µm cell strainer (Corning) and the strainer was washed with 1 ml and then 750 µl of buffer A. After the addition of Triton X-100 (final concentration 0.5%), the mixture was further homogenized with 50 strokes of the tight pestle (B). After filtering through a 40-µm strainer, nuclei were centrifuged (3,000 g, 5 min, 4 °C), resuspended in 1 ml of buffer B (320 mM sucrose, 10 mg ml -1 BSA, 3 mM CaCl 2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, 1 mM DTT, 1 × protease inhibitor, 0.12 U µl -1 RNaseIn, 0.06 U µl -1 SUPERasin) and purified using a 27% Percoll gradient solution. The Percoll mixture was centrifuged at...
Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and finely minced. Then, 2 g of the minced tissue was transferred to digestion buffer 1 (750 U ml -1 collagenase type 2 in 1 × PBS) and incubated at 37 °C in a water bath for 90 min. The partially digested tissue was collected by centrifugation (650 g, 5 min, 4 °C) and the pellet was resuspended in digestion buffer 2 (100 U ml -1 collagenase type 2, 2 U ml -1 dispase in PBS). After 30 min incubation at 37 °C in a water bath, the digestion was stopped by the addition of 2% FBS. Cells were filtered through a 100-µm and a 40-µm nylon strainer (BD Falcon), collected by centrifugation (650 g, 4 °C, 3 min) and washed with 1 × PBS, 2% FBS. Subsequently, a 20% Percoll gradient (15,000 g, 4 °C, 20 min) was used for cell purification. The layer containing cells was collected, washed in PBS containing 2% FBS, and viable cells were counted by Trypan Blue exclusion using a haemocytometer. Nuclei were profiled using a Chromium Controller (10X Genomics) according to the manufacturer's protocol.
Extended Data Fig. 10 Relevance for COVID-19 and GWAS studies. a, Global expression (log 2 FC) of ACE2 in all cardiac cells. b - d, Gene expression of ACE2, TMPRSS2, CTSB and CTSL in cardiomyocytes ( b ), FBs ( c ) and vascular cells ( d ). e, Multiplexed smFISH expression of DCN (cyan), KCNJ8 (green) and ACE2 (red), nuclei are DAPI-stained (dark blue) marking fibroblasts (#; expression of DCN ) and pericytes (*; co-expression of DCN and KCNJ8 ) in right ventricular tissue section. Scale bars, 5 µm. For statistics and reproducibility, see. f, The colour coding of the heat map shows the -log 10 ( P value) of the MAGMA GWAS enrichment analysis for the association between cell type-specific expression ( y axis) and GWAS signals ( x axis). The cell types refer to the subcluster annotations and GWAS studies refer to Supplementary Table. AF, atrial fibrillation; CAD, coronary artery disease; HF, heart failure; HR, heart rate; HT, hypertension; LVD, left ventricular diameter; NICM, non-ischaemic cardiomyopathy; PR, PR interval; PWAVE, P-wave duration; T2D, type 2 diabetes; QRS, QRS complex duration; QT, QT interval. Dots mark significant association...
Machine-readable layer
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{
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"name": "COVID-19 and GWAS disease relevance",
"text": "We define cells enriched for genes from 12 cardiovascular genome-wide association studies (GWAS) and involved in SARS-CoV-2 infection using MAGMA (Extended Data Fig. ). Atrial fibrillation GWAS signals are associated with transcriptional profiles in vCM3, owing to higher mean expression of CAV1, CAV2 and PRRX1. PR interval GWAS signals are associated with vCM3 and aCM5, with high expression of SCN5A, CAV1, ARHGAP24, MEIS1, TBX5 and TTN. GWAS signals for QRS duration are associated with specific gene expression in NC2 ( PRKCA, CEP85L, SLC35F1, SIPA1L1, KLF12 and FADS2 ). Coronary artery disease and hypertension GWAS signals are associated with transcripts from many cell lineages, particularly SMCs, FBs, and ECs, reflecting the relevance of vascular cells in both disorders."
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"name": "Research ethics for donor tissues",
"text": "Heart tissues (donors D1-D7 and D11) were processed at Wellcome Sanger Institute (Hinxton, UK) and obtained from deceased transplant organ donors after Research Ethics Committee approval (ref 15/EE/0152, East of England Cambridge South Research Ethics Committee) and informed consent from the donor families. Heart tissues (donors H2-H7) were processed at Harvard Medical School (Boston, Massachusetts, USA) and obtained from deceased organ donors after Human Research Ethics Board approval Pro00011739 (University of Alberta, Edmonton, Canada). Informed consent from donor families was acquired via the institutional Human Organ Procurement and Exchange Program (HOPE). Cardiovascular history was unremarkable for all donors (Supplementary Table )."
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"name": "Single nuclei isolation",
"text": "Single nuclei were obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl 2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1 × protease inhibitor, 0.4 U µl -1 RNaseIn, 0.2 U µl -1 SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40-µm cell strainer (Corning). After centrifugation (500 g, 5 min, 4 °C) the supernatant was removed and the pellet was resuspended in storage buffer (1 × PBS, 4% bovine serum albumin (BSA), 0.2 U µl -1 Protector RNaseIn). Nuclei were stained with NucBlue Live ReadyProbes Reagents (ThermoFis..."
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"position": 4,
"name": "Single-cell preparation",
"text": "Heart tissues (0.2-0.9 g) were transferred from cardioplegic solution into gentleMACS C-tubes (Miltenyi Biotec) containing enzymatic digestion base solution (100 µg ml -1 liberase TH Research grade and 50 µg ml -1 DNase I, HBSS 10 mM HEPES and 30 mM taurine). Tissues were minced using scissors (FST) and automatically digested using gentleMACS Octo Dissociator (Miltenyi Biotec) with heaters. Cardiomyocyte-depleted single-cell suspension were washed with base solution containing 20% fetal bovine serum (FBS) (Gibco), filtered through 70-µm nylon strainer (BD Falcon), collected by centrifugation (330 g, 10 min, 4 °C) and resuspended in base solution containing 0.2% FBS (Gibco). Cells were manually counted three times by Trypan blue exclusion after each centrifugation and resuspended at a concentration of at least 2 × 1..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "CD45 + cell enrichment",
"text": "Cell suspension was prepared as described above and subsequently labelled using anti-human CD45 monoclonal antibody-conjugated microbeads according to the manufacturer's protocol (Miltenyi Biotec). In brief, up to 10 7 cells were incubated for 15 min at 4 °C in 80 µl of PBS, BSA, EDTA buffer (1 × PBS pH 7.2, 0.5% BSA, 2 mM EDTA) containing 20 µl CD45 microbeads. Cell suspension was washed in PBS, BSA, EDTA buffer once and collected by centrifugation (330 g, 10 min, 4 °C). Resuspended cells were applied to MACS LS columns (Miltenyi Biotec). CD45-depleted cell fraction was discarded after three washes with PBS, BSA and EDTA buffer and the CD45 + cell fraction was collected in PBS, BSA and EDTA buffer by removal of the columns from the magnetic field. CD45 + cells were counted and resuspended in PBS, BSA and EDTA buffer to a concentration of\u0010..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Nuclei isolation for skeletal muscle",
"text": "Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and minced to obtain fragments of approximately 1 mm 3. Per sample, approximately 0.3 g of minced tissues was homogenized in 3 ml of buffer A (250 mM sucrose, 10 mg ml -1 BSA, 5 mM MgCl 2, 0.12 U µl -1 RNaseIn, 0.06 U µl -1 SUPERasIn, 1 × protease inhibitor) using Dounce tissue grinder set (Merck) with 50 strokes of the loose pestle (A). The homogenate was filtered through a 100-µm cell strainer (Corning) and the strainer was washed with 1 ml and then 750 µl of buffer A. After the addition of Triton X-100 (final concentration 0.5%), the mixture was further homogenized with 50 strokes of the tight pestle (B). After filtering through a 40-µm strainer, nuclei were centrifuged (3,000 g, 5 min, 4 °C), resuspended in 1 ml of buffer B (320 mM sucro..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Single-cell isolation for skeletal muscle",
"text": "Muscle tissue was washed in 1 × PBS, cleaned of any visible fat depositions and finely minced. Then, 2 g of the minced tissue was transferred to digestion buffer 1 (750 U ml -1 collagenase type 2 in 1 × PBS) and incubated at 37 °C in a water bath for 90 min. The partially digested tissue was collected by centrifugation (650 g, 5 min, 4 °C) and the pellet was resuspended in digestion buffer 2 (100 U ml -1 collagenase type 2, 2 U ml -1 dispase in PBS). After 30 min incubation at 37 °C in a water bath, the digestion was stopped by the addition of 2% FBS. Cells were filtered through a 100-µm and a 40-µm nylon strainer (BD Falcon), collected by centrifugation (650 g, 4 °C, 3 min) and washed with 1 × PBS, 2% FBS. Subsequently, a 20% Percoll gradient (15,000 g, 4 °C, 20 min) was used for ce..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Extended data figures and tables",
"text": "Extended Data Fig. 10 Relevance for COVID-19 and GWAS studies. a, Global expression (log 2 FC) of ACE2 in all cardiac cells. b - d, Gene expression of ACE2, TMPRSS2, CTSB and CTSL in cardiomyocytes ( b ), FBs ( c ) and vascular cells ( d ). e, Multiplexed smFISH expression of DCN (cyan), KCNJ8 (green) and ACE2 (red), nuclei are DAPI-stained (dark blue) marking fibroblasts (#; expression of DCN ) and pericytes (*; co-expression of DCN and KCNJ8 ) in right ventricular tissue section. Scale bars, 5 µm. For statistics and reproducibility, see. f, The colour coding of the heat map shows the -log 10 ( P value) of the MAGMA GWAS enrichment analysis for the association between cell type-specific expression ( y axis) and GWAS signals ( x axis). The cell types refer to the subcluster annotations and GWAS studies refer to Supplementary Table. AF, atrial fibri..."
}
],
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"@type": "HowToTool",
"name": "Main"
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"@type": "HowToTool",
"name": "Cardiomyocyte heterogeneity"
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{
"@type": "HowToTool",
"name": "Cardiomyocyte heterogeneity"
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{
"@type": "HowToTool",
"name": "Cardiac fibroblasts"
},
{
"@type": "HowToTool",
"name": "Cardiac fibroblasts"
},
{
"@type": "HowToTool",
"name": "Conduction system and neuronal cells"
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"name": "Single nuclei isolation"
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{
"@type": "HowToSupply",
"name": "Cardiomyocyte heterogeneity"
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{
"@type": "HowToSupply",
"name": "Conduction system and neuronal cells"
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"name": "Single nuclei isolation"
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{
"@type": "HowToSupply",
"name": "Single-cell preparation"
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{
"@type": "HowToSupply",
"name": "CD45 + cell enrichment"
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{
"@type": "HowToSupply",
"name": "Chromium 10X library preparation"
},
{
"@type": "HowToSupply",
"name": "Spatial validation using smFISH with RNAscope probes"
},
{
"@type": "HowToSupply",
"name": "Acquisition of skeletal muscle tissue"
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"headline": "Cells of the adult human heart",
"datePublished": "2020",
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"name": "Hongbo Zhang"
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