The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy.

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of βAR in isogenic hPSC-derived cardiomyocytes.

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (βAR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions.