RESUMO
Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the ß-isoform. We previously demonstrated that â¼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively ß-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than ß-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/ß-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted ß-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Miosinas , Cadeias Pesadas de Miosina/genética , IntegrinasRESUMO
Heart-forming organoids (HFOs) derived from human pluripotent stem cells (hPSCs) are a complex, highly structured in vitro model of early heart, foregut and vasculature development. The model represents a potent tool for various applications, including teratogenicity studies, gene function analysis and drug discovery. Here, we provide a detailed protocol describing how to form HFOs within 14 d. In an initial 4 d preculture period, hPSC aggregates are individually formed in a 96-well format and then Matrigel-embedded. Subsequently, the chemical WNT pathway modulators CHIR99021 and IWP2 are applied, inducing directed differentiation. This highly robust protocol can be used on many different hPSC lines and be combined with manipulation technologies such as gene targeting and drug testing. HFO formation can be assessed by numerous complementary methods, ranging from various imaging approaches to gene expression studies. Here, we highlight the flow cytometry-based analysis of individual HFOs, enabling the quantitative monitoring of lineage formation.