RESUMO
Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility with immunostaining.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Hidrogéis/química , Diferenciação Celular , Fibrina/metabolismo , Células Cultivadas , Técnicas de Cultura de Células/métodos , Células Estromais/citologia , Técnicas de Cultura de Tecidos/métodos , Técnicas de Cultura de Tecidos/instrumentaçãoRESUMO
Three-dimensional, human engineered heart tissue promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and provides a useful platform for in vitro cardiac development and disease modeling. This protocol describes the generation of fibrin-based engineered heart tissues (EHTs) containing hiPSC-CMs and human stromal cells. The platform makes use of racks of silicone posts that fit a standard 24-well dish. Stromal cells and hiPSC-CMs are cast in a fibrin hydrogel suspended between two silicone posts, forming an engineered tissue that generates synchronous contractions. The platform described herein is amenable to various measures of cardiac function including measurement of contractile force and calcium handling, as well as molecular biology assays and immunostaining.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Engenharia Tecidual , Fibrina , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , SiliconesRESUMO
COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.