Figure 3.
Semidirected differentiation into early embryonic germ lineages yields heterogeneous HORMAD1 and ZG16 expression. (A): Representative immunofluorescence staining with lineage-specific markers. Top Left: Embryonic stem cell (ESCs) and induced pluripotent stem cells (iPSCs) were differentiated into early mesoderm-like cells and stained for Brachyury (red). Top right: ESCs and iPSCs were differentiated into ectoderm-like cells and stained for PAX6 (green). Bottom: ESCs and iPSCs were differentiated into early endoderm-like cells and stained for SOX17 (red) (supplemental online Figs. 2, 3). Scale bars = 50 µm. (B): Gene expression analysis of HORMAD1 and ZG16 was assessed in each human ESC- and human iPSC-derived trilineage derivative across all lines for UI and I. Quantitative polymerase chain reaction (PCR) for HORMAD1 gene expression was found to be extremely low and variable across all lines, regardless of the induction of lineage (top). ZG16 expression varied in the directionality of differences in that some lines had significant increases on induction into each respective lineage (ectoderm: iPS1 and iPS2; endoderm: ES2, ES3, ES5, iPS3, and iPS6; mesoderm: iPS2, iPS3, and iPS4), but others showed a significant decrease (ectoderm: iPS6; endoderm: ES1; mesoderm: ES1 and ES4), although no clear correlation was found for certain lines or lineages (bottom). Error bars represent ±SD. All PCRs were run in triplicate. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ES, human embryonic stem cell line; iPS, human induced pluripotent stem cell line; I, induced; UI, uninduced.