Protocol for nuclear extraction from human heart tissue for single cell sequencing v1 (protocols.io.bcjqiumw)

A Method for Cryopreservation and Single Nucleus RNA-sequencing of Normal Adult Human Interventricular Septum Heart Tissue Reveals Cellular Diversity and Function

10.21203/rs.3.rs-136136/v1 ◽

2020 ◽

Author(s):

Amy Larson ◽

Michael T. Chin

Keyword(s):

Single Cell ◽

Human Heart ◽

Expression Patterns ◽

Interventricular Septum ◽

Cell Types ◽

Heart Tissue ◽

Gene Expression Patterns ◽

High Quality ◽

Normal Human ◽

Human Heart Tissue

Abstract Background: Single cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue. Methods: Using pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10x Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat. Results: Human heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes. Conclusions: Here we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

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A method for cryopreservation and single nucleus RNA-sequencing of normal adult human interventricular septum heart tissue reveals cellular diversity and function

BMC Medical Genomics ◽

10.1186/s12920-021-01011-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Amy Larson ◽

Michael T. Chin

Keyword(s):

Single Cell ◽

Human Heart ◽

Expression Patterns ◽

Interventricular Septum ◽

Cell Types ◽

Heart Tissue ◽

Gene Expression Patterns ◽

High Quality ◽

Normal Human ◽

Human Heart Tissue

Abstract Background Single cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue. Methods Using pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat. Results Human heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes. Conclusions Here we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

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Single Cell and Single Nuclei Analysis Human Heart Tissue v1 (protocols.io.veae3ae)

protocols.io ◽

10.17504/protocols.io.veae3ae ◽

2018 ◽

Cited By ~ 1

Author(s):

Monika Litvinukova ◽

Eric Lindberg ◽

Henrike Maatz ◽

Hongbo Zhang ◽

Michael Radke ◽

...

Keyword(s):

Single Cell ◽

Human Heart ◽

Heart Tissue ◽

Human Heart Tissue

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Benefit of microwave-assisted acid hydrolysis of proteins for mass spectrometric profiling of the human heart tissue proteome

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.3125 ◽

2007 ◽

Vol 21 (16) ◽

pp. 2779-2783 ◽

Cited By ~ 5

Author(s):

Mulu Gebremedhin ◽

Hongying Zhong ◽

Shaohua Wang ◽

Michael Weinfeld ◽

Liang Li

Keyword(s):

Acid Hydrolysis ◽

Human Heart ◽

Heart Tissue ◽

Mass Spectrometric ◽

Microwave Assisted ◽

Hydrolysis Of ◽

Tissue Proteome ◽

Human Heart Tissue

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IMMUNOLOGIC RELATION OF STREPTOCOCCAL AND TISSUE ANTIGENS

Journal of Experimental Medicine ◽

10.1084/jem.119.4.651 ◽

1964 ◽

Vol 119 (4) ◽

pp. 651-666 ◽

Cited By ~ 109

Author(s):

Melvin H. Kaplan ◽

Kathryn H. Svec

Keyword(s):

Human Heart ◽

Streptococcal Infection ◽

Heart Tissue ◽

Antigenic Determinants ◽

Heat Inactivation ◽

Prolonged Storage ◽

Acute Glomerulonephritis ◽

Streptococcal Antigen ◽

Reactive Antibody ◽

Human Heart Tissue

Sera from patients with recent streptococcal infection or non-suppurative sequelae exhibit with variable frequency a precipitin reaction in agar gel with a partially purified streptococcal antigen which has been shown previously to be immunologically related to human heart tissue. This precipitin could be absorbed from sera with normal human heart tissue homogenates but not with homogenates of other organs. Demonstration of this cross-reaction by heart absorption was found dependent both upon the serologic properties of individual sera and the nature or state of purification of the streptococcal product employed as test antigen. Evidence was obtained of a close association of heart-related and non-heart-related antigenic determinants in partially purified preparations of the streptococcal antigen by both gel diffusion and immunoelectrophoresis. On immunoelectrophoretic analysis, cross-reactive antigen exhibited a more rapid mobility toward the anode than M protein. It was destroyed by digestion with trypsin, pepsin, and chymotrypsin. Based on specific absorption tests with a Type 5 and Type 19 strain, the antigen was localized to cell walls and to a lesser extent to cell membranes of these strains. Precipitating activity related to cross-reactive antibody was localized to the immunoglobulin zone in immunoelectrophoresis. Reactive sera showed diminution or loss of serological activity following heat inactivation at 56°C or after prolonged storage at 4°C. Sera containing cross-reactive precipitating antibody exhibited an immunofluorescent reaction with sarcolemma of cardiac myofibers, which was inhibited by streptococcal cross-reactive antigen. By this inhibition test, the immunofluorescent reaction related to cross-reactive antibody could be distinguished from that due to other heart-reactive factors. Antibody to streptococcal cross-reactive antigen defined by precipitation-absorption tests was observed in 24 per cent of patients with recent history of uncomplicated streptococcal infection and in the majority of patients with acute rheumatic fever, rheumatic heart disease, or acute glomerulonephritis. It was observed rarely in patients with non-streptococcal related disease. These data provide evidence that induction of cross-reactive autoantibody to heart in certain individuals is associated with streptococcal infection.

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