Selection of Human Myocardial Precursors Figure 2. Sorted aMHC-GFP+ hESCs form CMs in vitro. aMHC-GFP hESCs were cultured under differentiation conditions for 8 days. (A) Day 8 hEBs were suspended as single cells and sorted for GFP expression. Propidium iodide was used to discriminate live versus dead cells. A typical sort is shown. (B) GFP+ cells were re-cultured under differentiation conditions for an additional 6 days, then analyzed in situ by indirect immunofluorescence for expression of cTnT and cardiac a-actinin. Cells were counterstained with DAPI to identify nuclei. Typical micrographs are shown. Expression of cTnT and cardiac a-actinin was seen throughout the culture, which demonstrated multiple foci of spontaneous contractile activity (Supplementary Videos 1 and 2). Bar, 400 mm. doi:10.1371/journal.pone.0016004.g002 line, we analyzed cells for single chromosomal region gains and losses by array comparative genomic hybridization [17]. This showed that aMHC-GFP hESCs maintain a normal karyotype at the level of individual gene regions (Fig. 4). The aMHC-GFP reporter identifies an early myocardial precursor that gives rise to multiple CM subtypes Others have shown that CMs derived from hESCs in culture can display the molecular and electrical properties of embryonic ventricular, atrial and nodal tissue [6]. This heterogeneity occurs with differentiation in culture using a variety of conditions [7,8,9,10], and suggests that hESCs are capable of giving rise to myocardial precusors that precede the developmental branchpoint between first and second heart field specification. We counted the number of GFP+ (aMHC + ) hESCs over the course of hEB differentiation, and found that these cells started to appear in culture at day 5, commensurate with the onset of GATA4 and NKX2-5 expression, and several days before the expression of cTnT, which is associated with spontaneous contractile activity [18] (Fig. 5). Since the reporter appeared to identify early human myocardial precursors (hMPs), we wanted to determine whether these hMPs PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16004