To examine the proliferative and differentiative capacities

To examine the proliferative and differentiative capacities of the GSpos and GSVpos populations, we performed limit dilution analysis to determine the clonogenic frequency of single flow sorted Nocodazole Supplier (Elliott et al., 2011). Under these adherent culture conditions, the GSpos cells had a clonal frequency of 0.8% (~1/123) whilst 0.3% (~1/331) of the GSVpos cells gave rise to clones (Fig. 3E). Neither fraction gave rise to spontaneously contractile colonies, suggesting that d10 NKX2-5pos CMs lack the capacity for either further proliferation and/or differentiation in these conditions. In order to assess the potential of these cells to proliferate and differentiate, we used a clonal assay in which single cells were seeded into each well of a 96 well plate. Analysis of clones grown from single GSpos cells showed that of the 14 colonies (from 2784 cells deposited) 11 expressed the smooth muscle marker Calponin (CNN1), 2 were CNN1+TNNT2+ and 1 was TNNT2+ (Figs. 3F–H). To ascertain if the GSVpos population was derived from the GSpos population, cells representing each population were flow sorted, re-aggregated and cultured as contractile cardiospheres (Figs. 4A, B; Supplementary Video 1). After 4days, the majority of GSpos cells had become GSVpos (75±7.2%; n=3) whilst almost all GSVpos cells retained marker expression (Figs. 4C, D). Taken together these data support the hypothesis that GSpos (i.e. NKX2-5posSIRPApos) precursors display greater proliferative capacity and can form smooth muscle cells and CMs whilst GSVpos (i.e. NKX2-5posSIRPAposVCAM1pos) cells consist mainly of committed CMs (Fig. 4E).
Discussion The cell surface markers SIRPA, VCAM1 and CD34, were used to isolate and characterize cardiac precursor populations of the endothelial and myogenic lineages (Fig. 4E). The NKX2-5posCD34pos fraction corresponded to emerging cardiac endothelial precursors, defined by the transient expression of NKX2-5 during their early development. NKX2-5posSIRPAposVCAM1pos cardiomyocytes arose from an NKX2-5posSIRPApos intermediate, demonstrating a lineage relationship between these two cell populations and establishing VCAM1 up-regulation as a key marker of cardiomyogenic commitment. Further, recent work suggests that VCAM1 has utility as a definitive cardiomyocyte marker in mice (Pontén et al., 2013). At present, it is difficult to correlate our in vitro findings with the in vivo expression pattern of these markers since those studies require human embryos between 3 and 4weeks of gestation. We have previously shown that transcripts for both SIRPA and VCAM1 are expressed at high levels in foetal hearts at 9weeks suggesting that these markers are expressed in vivo (Elliott et al., 2011). Nevertheless, given that mouse models are inadequate for establishing human protein expression profiles, as demonstrated for c-KIT here and previously for SIRPA (Dubois et al., 2011), obtaining experimental data from early human embryos is a future priority. Molecular markers permitting the identification and isolation of distinct cardiogenic cell populations will be useful for understanding the developmental logic of human cardiogenesis, pharmaceutical screening and regenerative medicine (Burridge et al., 2012; Sturzu and Wu, 2011; Thavandiran et al., 2013; Tiscornia et al., 2011). In this context, the temporal and contingent relationships of the cell lineage markers described here form a foundation for placing newly identified markers within the cardiovascular lineage fate map. Subsequently, specific marker combinations could be used to isolate and study, in a manner analogous to the haematopoietic system, homogenous, well-defined, cardiac cell populations. The following are the supplementary data related to this article.
Acknowledgments We thank the Australian National Health and Medical Research Council (NHMRC) (DAE; 606586, Fellowship AGE, EGS) the NHMRC\'s Independent Research Institute Infrastructure Support Scheme, the Victorian Government\'s Operational Infrastructure Support Program, the Qatar National Research Foundation (MC, ART, AGE, EGS & DAE), JDRF (EGS & AGE) and the Netherlands Institute of Regenerative Medicine (RPD & CLM). Australian Post-graduate Awards (RJPS, BWF) and an EU Marie Curie International Outgoing Fellowship (DJA).