A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity

Abstract

Aims Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs. Methods and results We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia. Conclusion We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.

Androgen Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men

“…Cell culture and differentiation. H9 human embryonic stem cells were maintained and differentiated as described previously 10,12 . Briefly, human embryonic stem cells (hESCs) were maintained in mTeSR medium.…”

“…Given the highly significant association of poor outcomes in COVID-19 patients with cardiovascular complications 2,3 and the significant role of ACE2 in cardiac physiology 16 , we chose to focus on the regulation of ACE2 levels in cardiac cells. Due to limitations associated with isolation and maintenance of human cells from primary tissue, we used our previously established hESC differentiation method as an alternative strategy to generate cardiac cells (Supplementary Figure 1B) 12 Previously published transcriptomics data on hESC-derived cells generated using this method 12 confirmed the expression of SARS-CoV-2 receptor and co-receptors mRNAs in cardiomyocytes and non-cardiomyocyte (Supplementary Figure 1C). The differentiated cells also stained positive for ACE2 as assessed by immunofluorescence imaging (Supplementary Figure 1D).…”

“…The bulk RNA sequencing dataset analyzed here was previously reported 12 . Briefly, human pluripotent stem cell (H9 with knock-in MYH6:mCherry reporter)-derived cardiomyocytes were prepared as described previously 10,12 . Reporter tagged cardiomyocytes were isolated from the negative fraction of the culture by FACS sorting.…”

“…To identify candidate drugs capable of modulating ACE2 protein levels, we took advantage of our previously established methods to generate cardiac cells from human embryonic stem cells at large scale [10][11][12] and performed a high content screen with a library of 1443 FDA-approved drugs and a subsequent in silico screen with the ZINC15 library of over 9 million drug-like compounds. We discovered that drugs most effective in reducing ACE2 protein levels converge on androgen receptor (AR) signaling inhibition as a common mechanism of action.…”

SARS-CoV-2 Infected Cardiomyocytes Recruit Monocytes by Secreting CCL2

“…Our previous studies showed that hPSC-derived CMs are permissive to SARS-CoV-2 infection 10 , and established a platform to model response of CMs to SARS-CoV-2 infection. CMs were derived from an MYH6:mCherry H9 hESC reporter line or a human iPSC line 18 (Extended Data Fig. 1a).…”