Selection of Human Myocardial Precursors Figure 3. hMPs form muscle in vivo. Teratomas formed from aMHC-GFP hESCs by renal capsule grafting in SCID mice were analyzed by hematoxylin and eosin staining to identify tissue structures (left), and immunohistochemistry with anti-GFP antibody (right) to identify tissues arising from hMPs. Section from typical teratoma shown (n = 7). Primitive neural epithelium (star) and glandular intestinal structures (arrowheads) can be seen surrounding striated muscle (arrows) (left), which stains positive for GFP expression (brown; right). GFP was expressed exclusively in mononucleated myofibers within the teratomas, and was not found in any other tissues (not shown). Bar, 100 mm. doi:10.1371/journal.pone.0016004.g003 were capable of generating a heterogeneous population of human CMs based on molecular and electrical characteristics. We differentiated aMHC-GFP hESCs for 21 days, and manually dissected GFP+ hEBs for analysis by qPCR and microelectrode array. Evaluation of individual GFP+ hEBs by qPCR demonstrat- ed that molecular signatures characteristic of embryonic atrial (Islet-1 (ISL-1)+ sarcolipin (SLN)+ Na+/K+ hyperpolarization- activated cyclic nucleotide-gated channel 4 (HCN4)2 myosin light chain-2 atrial (MLC2a)+ MLC2v2 aMHC+ cTnT+), left (ISL-12 SLN2 HCN42 MLC2a2 MLC2v+ aMHC+ cTnT+) and right (ISL-1+ SLN2 HCN42 MLC2a2 MLC2v+ aMHC+ cTnT+) ventricular, and nodal (ISL-1+ SLN2 HCN4+ MLC2a2 MLC2v2 aMHC+ cTnT2) tissue were present in distinct hEBs (Fig. 6A) at an approximate ratio of 40% ISL-1+ SLN+ HCN42 MLC2a+ MLC2v2 aMHC+ cTnT+, 10% ISL-1+ SLN2 HCN4+ MLC2a2 MLC2v2 aMHC+ cTnT2, 30% ISL-1+ SLN2 HCN42 MLC2a2 MLC2v+ aMHC+ cTnT+ and 20% ISL-12 SLN2 HCN42 MLC2a2 MLC2v+ aMHC+ cTnT+ among .30 hEBs analyzed. To determine whether the electrical properties of GFP+ hEBs were similarly heterogeneous in nature, we allowed dissected hEBs to adhere to microelectrode arrays (MEAs) (Fig. 6B) and performed simultaneous recording of extracellular field potentials Figure 4. The aMHC-GFP reporter line maintains genomic integrity. aMHC-GFP hESCs (H9; 46,XX) passaged ten times after stable selection were analyzed by array comparative genomic hybridization. The log2 ratios for all loci on the array were plotted in genome order from chromosome 1pter to Xqter. The data were normalized so that the log2 ratio = 0 for genomic regions that are present in diploid copy number. Male reference DNA was used in the hybridization as indicated by the sex mismatch for chromosome X (arrow). There was no evidence of copy loss or gain, consistent with maintenance of euploidy. doi:10.1371/journal.pone.0016004.g004 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16004