SUPPLEMENTARY INFORMATION

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1 Suppl. Fig. 1 in vivo expression of ISL1 in the human fetal heart. a, Hematoxylin eosin staining showing structures of left atrium and left atrium appendage (*) of a human fetal heart at 11 weeks of gestation. Panels on the right showing immohistochemical analyses for the LA with indicated antibodies. b, Immunostaining of sections from RA region with indicated antibodies. Yellow arrows pointing cells stained positive for ISL1 and PECAM1. RA; right atrium, LA: left atrium, RV: right ventricle. Scale bars: 25 µm (a,b) and 50 µm in far right panels in b. 1

2 Suppl. Fig. 1 in vivo expression of ISL1 in the human fetal heart (continued). c, d, Immunostaining of adjacent sections from human fetal hearts at 11 weeks (c) and 18 weeks (d) of gestation with indicated antibodies. A same field of each section is presented from a lower (left panel) to a higher (middle panel) magnification. RA; right atrium, SVC: Super vena cava, RCA: right coronary artery, AVC: atrioventricular canal, RV: right ventricle. Scale bars: 25 µm. 2

3 Suppl. Fig. 2 Generation of transgenic and knock-in hes cell lines. a, A diagram of the human ISL1-βgeo BAC transgenic construct. A βgeo cassette and an FRT-flanked antibiotics cassette are inserted into ISL1 endogenous translation start site. b, Cells dissociated from day 6 EBs of the human ISL1-cre knock-in line were stained positive for cre recombinase and ISL1 after a two-day co-culture with feeders. Scale bar: 5 µm. c, A scheme of the human ISL1-cre DsRed lineage tracing strategy in hes cell model system. In order to reduce potential effects on the regulatory sequences of the ISL1 locus, the FRT-flanked antibiotics cassette in knock-in locus was excised by transient transfection of an Flpase expressing plasmid. d, A typical beating human ISL1-cre DsRed EB on a gelatin-coated cell culture plate containing a cluster of DsRed+ cells. 3

4 Suppl. Fig. 3 Gene expression of the human ISL1-cre DsRed ES cells and KDR staining in the human fetal hearts. a, FACS analysis on cells dissociated from day 8 EBs of human ISL1-cre DsRed cells and stained with FITC-conjugated anti-kdr antibody. b, RT-PCR showing gene expression of DsRed+ and DsRed- cells isolated from day 8 EBs of human ISL1-cre DsRed cells. c, d, Cross sections of 18 weeks (c) and 11 weeks (d) of gestation human fetal heart co-stained for ISL1 and KDR in the RA areas, showing that some ISL1- cells are KDR+ and some ISL1+ cells stained negative for KDR. RA; right atrium, LA, left atrium, RCA: right coronary artery, AVC: atrioventricular canal, RV: right ventricle, IAS: intra atrial septum. 4

5 Suppl. Fig. 4 Single cell derived clones developed on MEF feeders. To confirm that the DsRed+ colonies studied in the clonal assays are derived from single cells, we generated an additional transgenic pcag-flox-egfp ES line in the human ISL1-cre knock-in background. DsRed+ and egfp+ cells were purified from day 8 EBs. Equal numbers of red and green cells were mixed and plated at up to a 4-fold density used in the clonal assay. a, Distinct colonies from mixed cultures showing either DsRed or egfp expressing exclusively after 10 days of co-culture. Scale bars: 10 µm. b, 101 colonies/clusters were formed from DsRed+ and egfp+ cells and are summarized. c, The number of colonies developed on MEF feeders showing an arithmetically linear relationship to the numbers of input cells. Bars represent mean ± s.d.; n=3. 5

6 Suppl. Fig. 5 Expansion and characterization of hes cell-derived ISL1+ cardiac progenitors. a, ISL1+ cells increased by BIO treatment. Day 6 EBs of human ISL1-βgeo BAC transgenic cells were dissociated and cultured on mouse CMC feeders for two days before treated with and without BIO for another five days. Colonies were stained with anti-isl1 antibody and counted for ISL1+ ones. b, Immunostaining of a typical BIO-treated ISL1+ colony. c, qpcr showing the gene expression of Wnt3a-expanded DsRed+ cells. Cells dissociated from day 7 EBs of human ISL1-cre DsRed cells were plated on Wnt3a-secreting feeders, cultured for five days and FACS isolated. Gene expression of DsRed+ cells was compared with DsRed- cells. Bars represent mean ± s.d.; n=3. 6

7 Supplementary Table 1. Molecular markers of single DsRed+ cell-derived progenitor clones. The total 95 tested clones are categorized into four groups based on their expression pattern of ISL1 and NKX2-5. The percentage of each group is listed under the group definition. Typical clones of each group are presented with gene expression pattern as well as their total numbers. 7