Supplementary MaterialsDocument S1. GFP through the locus. This dual atrial NKX2.5EGFP/+-COUP-TFIImCherry/+ reporter line allowed identification and selection of GFP+ (G+)/mCherry+ (M+) CMs following cardiac differentiation. These cells exhibited transcriptional and functional properties of atrial CMs, whereas G+/M? CMs displayed ventricular characteristics. Via CRISPR/Cas9-mediated knockout, we demonstrated that COUP-TFII is not required for atrial specification in hPSCs. This new tool allowed selection of human atrial and ventricular CMs from mixed populations, of relevance Batefenterol for studying cardiac specification, developing human atrial disease models, and examining distinct effects of drugs on the atrium versus ventricle. but also in pre-clinical drug testing and safety pharmacology (Beqqali et?al., 2009, Braam et?al., 2010, Maddah et?al., 2015, van Meer et?al., 2016, Sala et?al., 2016). Despite substantial improvements in the efficiency of hPSC differentiation to CMs during the last decade, the majority of directed cardiac differentiation protocols yield heterogeneous CM populations, largely composed of ventricular CMs (Mummery et?al., 2012). Recently, we demonstrated efficient generation of atrial CMs from human embryonic stem cells (hESCs) (Devalla et?al., 2015). These hESC-derived atrial CMs (hESC-AM), resemble human fetal atrial CMs at the molecular and functional level and have already DPP4 proven to be a predictive and reliable pre-clinical model for atrial selective pharmacology (Devalla et?al., 2015). Although hESC-AMs represented the majority of CMs (approximately 85%) in our directed differentiation protocol, other cardiac subtypes (mostly ventricular CMs with less than 1% of nodal cells) were also present. To select hESC-AM populations and study their differentiation (Tsai and Tsai, 1997). During murine heart development, COUP-TFII is first detected in the visceral mesoderm and sinus venosus, then progresses to the common atrium, and becomes restricted to CMs of the atrial chambers at later stages of development (Pereira et?al., 1999, Wu et?al., 2013). This indicates that COUP-TFII is an important atrial-enriched transcription factor and prompted us to develop an atrial hESC Batefenterol reporter line using CRISPR/Cas9 genome-editing technology to insert sequences encoding the red-fluorescent protein mCherry into one allele of the genomic locus. Since COUP-TFII expression is not confined to CMs, but is also expressed in other mesodermal cell types (for example, venous endothelial cells, skeletal muscle, and kidneys) (Lee et?al., 2004, You et?al., 2005, Yu et?al., 2012), as well as endodermal (for example, liver and pancreas) (Zhang et?al., 2002) and some ectodermal derivatives (cerebellum, eye, and ear) (Kim et?al., 2009, Tang et?al., 2010, Tang et?al., 2005), we chose the well-established human cardiac NKX2.5EGFP/+ reporter (Elliott et?al., 2011) to develop a unique dual reporter line that would allow identification and purification of hESC-AMs. Transcriptional and functional analysis of sorted GFP+ (G+)/mCherry+ (M+) double-positive CMs clearly demonstrated their atrial identity, whereas G+/M? CMs belonged to the ventricular lineage. In addition, we found that complete loss of COUP-TFII did not affect the differentiation Batefenterol toward AMs, based on both molecular and functional analysis. Purification of hESC-AMs will likely be important for Batefenterol optimization and standardization of assays in cardiac drug screening and modeling atrial diseases, such as atrial fibrillation, and understanding underlying molecular mechanisms for atrial specification and disease. Results Generation of a Fluorescent Dual Reporter by CRISPR/CAS9-Mediated Targeting of COUP-TFII in hESC-NKX2.5-GFP To generate an atrial hESC reporter line, we inserted sequences encoding the red fluorophore mCherry into the genomic locus of ((sgRNA 1 and 2) (Figure?1A). NKX-GFP hESCs were transfected with the COUP-TFII-mCherry targeting vector and one of the sgRNAs co-expressed from the Cas9 vector (Figures 1B and 1C). After antibiotic selection, the excision of the blasticidin-resistance gene was mediated using flippase site-specific recombination (Figure?1C). Correctly targeted clones displayed a 0.8 kb PCR product following screening of the 5 end and a 2.9 kb product (1.7 kb after blasticidin excision) of the 3 end (Figure?1D). Following clonal selection by fluorescence-activated cell sorting (FACS), correct targeting of the subclones as well as excision of the blasticidin-resistance cassette was reconfirmed by PCR. In addition, a PCR screen was performed to determine whether mCherry was inserted into one or both alleles (Figure?1D). For subclones in which mCherry was monoallelic targeted, Batefenterol the genomic integrity of the wild-type (WT) allele was confirmed by Sanger sequencing of the PCR product. Following sequencing, we identified that, in all mCherry monoallelic-targeted clones from sgRNA 1 and 2, the other allele possessed.