Flow cytometry analysis showed that

Flow cytometry analysis showed that independent targeted clones expressed remarkably similar levels of the inserted reporter genes, highlighting the consistency of expression produced by this vector configuration (Figure 1C). This consistency also enabled us to conclude that all the selectable marker genes adversely affected expression of the upstream fluorescent reporter (Figure S1F). Intracellular flow cytometry of GT-Luc2 H9 cells showed that independent clones also expressed Luc2 at consistent levels while transplantation experiments indicated expression was sufficient to observe subcutaneously grafted cells using 6XHis imaging (Figure 1D). Similarly, surface expression of GLuc could be detected on GT-GLuc iPSCs using flow cytometry, and consistent levels of GLuc activity could be readily assayed on live cells (Figure 1E). We examined the reliability of expression during the differentiation to derivatives of the three embryonic germ layers, mesoderm, endoderm, and ectoderm. These studies indicated that robust expression was maintained when GT-GFP H9 hESCs or GT-tdTomato iPSCs were differentiated to hematopoietic mesoderm, with readily detectable expression in individual hematopoietic cells by fluorescence microscopy and flow cytometry (Figures 2A, 2B, and S2A). Similarly, tdTomato expression from the GAPDH locus (GT-TdTom) was maintained at robust levels in cardiac mesoderm at differentiation day 7, identified by expression of GFP from the NKX2-5 locus (Elliott et al., 2011) (Figure 2C). Bright GT-tdTomato expression was also observed in CXCR4+EpCAM+ definitive endoderm cells at differentiation day 4 (Figures 2D and S2B), with expression levels similar to that seen in undifferentiated GT-tdTomato PSCs (Figure 2E). Using a neural differentiation protocol, we observed strong and consistent tdTomato expression in tyrosine hydroxylase (TH)-positive ventral midbrain dopamine neurons at differentiation day 30 (Figure 2F, see also GT-GFP expression in Figure S2C). To further explore the spectrum of cell types that expressed the GT-reporter system, we generated teratomas from GT-lacZ PSCs by subcutaneous injection of 106 undifferentiated cells into NOD/SCID/IL2Rγ null mice (Shultz et al., 2005). After 2–3 months, palpable teratoma masses were removed, fixed in paraformaldehyde, and subsequently stained with X-gal to reveal cells expressing β-galactosidase activity. This analysis revealed that the surface of teratomas and the surface of dissected teratoma subfragments stained strongly and uniformly with X-gal (Figure S2D). Examination of eosin-stained sections revealed a variety of differentiated tissue types, including cartilage (Figure 2G), epithelium containing goblet-like cells and surrounding mesenchyme (Figure 2H), and pigmented epithelium (Figure 2I) that displayed strong X-gal staining, suggesting the GT-lacZ transgene continued to be expressed, even after an extended period of differentiation in vivo.
Discussion We have generated a series of vectors designated GAPTrap, or GT, based on the GAPDH locus, a ubiquitously and robustly expressed housekeeping gene. We have constructed and tested a range of reporters and have shown that their expression is maintained in a wide variety of cell types, and for longer than 2 months in vivo, making the system ideal for the long-term tracking of cells in vitro and subsequent transplantation. In addition to commonly used fluorescent proteins, we have tested a number of other reporters we believe will be useful for in vivo cell tracking. For example, luciferase (Luc2) allows the visualization of cells in living animals using bioluminescent imaging. As stem cell technologies transit the preclinical testing phase of development, such imaging systems will be useful for understanding the behavior of differentiated cells in animal models. We also found that a previously described surface-directed luciferase, GLuc (Santos et al., 2009), was robustly expressed, despite the fact that the signal sequence required for trafficking to the cell membrane was C-terminal of cytoplasmic GAPDH. Lastly, we have illustrated how a GT-lacZ expression system enables the simple detection of PSC-derived cells in a context compatible with standard paraffin-embedded histological sections.