Blasticidin and Puromycin resistance cassettes for drug selection of undifferentiated stem cells and functional hESC-derived cardiomyocytes. Additional vectors were constructed to produce hESC lines with eGFP and mCherry fluorescent reporters of mesoderm and cardiac lineages and also fluorescent Ginsenoside-Ro Histone2B fusion proteins that allow real-time sensing of the DNA content and recognition by automated algorithms for cell screening and tracking. We used 3 criteria to validate that the isolation procedure did not adversely affect the cardiomyocytes, indicati that the drug-isolated cardiomyocytes were physiologically normal. We describe single cell cloning and drug selection procedures methods and a suite of lentiviral vectors to engineer hESC lines for visualization, tracking and purification of pluripotent stem cells and their differentiated cardiomyocyte derivatives. These methods overcome variegation and downregulation of fluorescent reporters and other markers observed without enrichment. The FACS and cloning protocols yielded homogeneous levels of fluorescent reporter protein expression sufficient for automated tracking and quantitative analyses, such as biosensors of DNA content, and the protocols and vectors could be broadly applied to biosensors of subcellular structures or signal transduction pathways as well as improving the identification of hESC derivatives after engraftment in animal models of disease or regeneration. Although the experiments shown were performed using H9 cells, similar outcomes were obtained with the PGKH2B fluorescent protein vectors, Rex- and aMHC-driven fluorescent and selectable markers using HUES13 and human induced pluripotent stem cells derived in our laboratory. Dual cassette lentiviral vectors were developed to enable drug selection of pluripotent stem cells with G418 or Blasticidin and cardiomyocytes with Puromycin. The two-stage selection procedure first eliminated feeder and Cinoxacin spontaneously differentiating cells, which in our hands compromise cardiomyocyte differentiation in EBs. Puromycin selection yielded greater than 96% pure cardiomyocytes at or after day 12. We exploited the purification procedure to profile hESC-derived cardiomyocytes gene expression with the finding that their profile strongly resembled that of the adult human heart. Additionally, the selected cardiomyocytes exhibited action potentials that resemble human fetal cardiomyocytes.