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

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.

Current trends in the creation of cell-free allo- and xenotissues for reconstruction of heart structures

Tissue engineering has significant potential for solving the problems of durability of biological tissues when used in cardiac and vascular reconstructive surgery. A decellularization technology has been proposed for obtaining a biomaterial, morphologically and functionally similar to the damaged human heart tissue. This review discusses various aspects and models of biological tissue decellularization, including the modern technology of using supercritical carbon dioxide as the most eco-friendly and promising method.

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

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.

Abstract MP143: Harnessing Glycomics to Understand Cardiac Biology and Disease

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function, and the glycan-moiety attached to a protein is critical for proper protein folding, stability, and signaling. Despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. Here, we used an innovative mass spectrometry approach to generate the first glycan structure libraries for primary human heart tissue, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM), containing >260 N- and O- glycans. Comparing the glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, 21 structures significantly differed, and the high mannose class is increased in enriched CM. Moreover, >30% of the glycome significantly changed across 20-100 days of in vitro differentiation, and only 23% of the N -glycan structures were shared between hiPSC-CM and primary CM. Overall, these observations are an important complement to genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. These data are also expected to aid in the evaluation of the immunogenic potential of hiPSC-CM for transplantation. Finally, harnessing differences observed between immature, proliferative hiPSC-CM and adult primary CM may be exploited to drive in vitro differentiation towards a more mature phenotype. Building on these data, current efforts are underway to develop chamber- and cell-type specific views ( e.g. cardiomyocytes, fibroblasts) of the glycome in the healthy and failing human heart. Such analyses provide a key link to understand the role glycosylation plays in cell-type specific functions and cardiac disease. The structural differences observed here, either among cell types or stages of differentiation, require complex regulation of multiple enzymes in the biosynthetic pathway, and therefore would be challenging to measure with antibody arrays, RNAseq, or proteomics. Therefore, continued application of structure-based glycomics approaches, such as the method used here, will be essential for elucidating the roles that glycans and glycoproteins play during developmental and disease processes in the human heart.

Human CardioChimeras: Creation of a Novel ‘Next Generation’ Cardiac Cell

BackgroundCardioChimeras (CCs) produced by fusion of murine c-kit+ cardiac interstitial cells (cCIC) with mesenchymal stem cells (MSCs) promote superior structural and functional recovery in a mouse model of myocardial infarction (MI) compared to either precursor cell alone or in combination. Creation of human CardioChimeras (hCC) represents the next step in translational development of this novel cell type, but new challenges arise when working with cCICs isolated and expanded from human heart tissue samples. The objective of the study was to establish a reliable cell fusion protocol for consistent optimized creation of hCCs and characterize fundamental hCC properties.Methods and ResultsCell fusion was induced by incubating human cCICs and MSCs at a 2:1 ratio with inactivated Sendai virus. Hybrid cells were sorted into 96-well microplates for clonal expansion to derive unique cloned hCCs, which were then characterized for various cellular and molecular properties. hCCs exhibited enhanced survival relative to the parent cells and promoted cardiomyocyte survival in response to serum deprivation in vitro.ConclusionsThe generation of hCC is demonstrated and validated in this study, representing the next step toward implementation of a novel cell product for therapeutic development. Feasibility of creating human hybrid cells prompts consideration of multiple possibilities to create novel chimeric cells derived from cells with desirable traits to promote healing in pathologically damaged myocardium.Clinical Perspective“Next generation” cell therapeutics will build upon initial findings that demonstrate enhanced reparative action of combining distinct cell types for treatment of cardiomyopathic injury.Differential biological properties of various cell types are challenging for optimization of delivery, engraftment, persistence, and synergistic action when used in combination.Creation of a novel hybrid cell called a CardioChimera overcomes limitations inherent to use of multiple cell types.CardioChimeras exhibit unique properties relative to either parental cell anticipated to be advantageous in cellular therapeutic applications.CardioChimeras have now been created and characterized using cells derived from human heart tissue, advancing initial proof of concept previously demonstrated with mice.CardioChimeras represent an engineered solution that can be implemented as a path forward for improving the outcome of myocardial cell therapy.

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, 21 structures significantly differed, and the high mannose class is increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.