Mouse nhpESC chimeras were also prepared using

Mouse–nhpESC chimeras were also prepared using the mouse tetraploid complementation assay (Nagy, 2003). We electrofused mouse 2-cell embryos to produce 4N embryos and permitted these to develop to the expanded blastocyst stage before injecting them with GFP-expressing nhpESCs and performing embryo transfers to ICR pseudopregnant recipients. Harvest of fetal material around E12.5 showed high implantation sites with chimera prepared with nhpESC 2706 ESCs (Table 1, column e), but no normal fetal development and mostly necrotic or reabsorbing implantation sites upon sacrifice (Table 1, column h). To analyze mouse–nhpESC chimeras at the cellular level, a few normal E12.5 fetuses were selected for immunohistochemical analysis (Fig. S5). We counterstained 10μm sections with anti-GFP antibody to compare with any detected GFP expression. Preliminary analysis of ectoderm (spinal cord tissue: Fig. S5A1), mesoderm (pericardial tissue: Fig. S5B1) and endoderm (urogenital tissue; Fig. S5C1) layers in an interspecies fetus suggested extensive survival of GFP-expressing cells that co-localized precisely with anti-GFP staining (Fig. S5A2, S5B2, and S5C2). However, control tissue sections from a fertilized E12.5day mouse embryo suggested extensive autofluorescence following fluorescein and rhodamine excitation in a variety of tissues, rendering fluorescent analysis unreliable (fluorescein excitation: Fig. S5A3, S5B3, and S5C3; rhodamine excitation: Figs. S5A4, S5B4, and S5C4). Efforts to control for endogenous fluorescence by using various blocking agents prior to application of primary and secondary ccr5 antagonist were not successful (our unpublished data). On selected interspecies embryos produced with GFP-expressing rhesus nhp2706 ESCs (a male line), we explored if SRY and GFP DNA could be detected by PCR analysis (Fig. 4). For a positive control, we used DNA isolated from a transgenic male monkey carrying the GFP transgene (ANDi) (Chan et al., 2000), demonstrating the detection of SRY DNA (Fig. 4, lane 3) and GFP DNA (Fig. 4, lane 14). However, no DNA from the embryonic tissues of these interspecies chimeras produced positive bands with primers from either SRY (Fig. 4, lanes 4–6) or GFP (Fig. 4, lanes 9–13), suggesting that no nhpESCs had survived in the developing mouse fetuses. Analysis of an interspecies chimeric blastocyst outgrowth produced with GFP-expressing nhp2706 ESCs also did not detect SRY DNA after 1month in culture (Fig. 4, lane 2). This particular colony did not demonstrate GFP expression in surviving cells after a few days of culturing in vitro. Finally, we investigated GFP-expressing nhpESC survival, proliferation and integration with mouse cells in vitro following blastocyst outgrowth on sterile coverslips (Fig. 5). Chimeric injection blastocysts outgrown for 3days showed that GFP-expressing cells remained largely clustered together without significant intermixing with mouse cells (Fig. 5A–C). Likewise, aggregation chimeric blastocyst outgrown for 17days in vitro (Fig. 5D–F) demonstrated that while the GFP-expressing nhpESCs proliferated over the 2weeks in culture, the nhpESCs did not integrate into the mouse ICM cellular area (Fig. 5D–F, * indicates mouse differentiated cells derived from the mouse ICM). Regardless, the survival of pluripotent, GFP expressing nhpESCs in mouse chimeric blastocyst outgrowths were low (~2.5%) overall.
Discussion The contribution of ESCs and other PSCs to chimeric offspring resulting in germ-line transmission is the most stringent assay for demonstrating biological pluripotency (reviewed by Behringer, 2007). This chimera assay has resulted in significant insights into the various categories of PSCs, even with mice, since embryonal carcinoma, embryonic germ, ESCs and PSCs generated by induced pluripotency all pass this test, whereas stem cells from epiblasts do not (Tesar et al., 2007; Brons et al., 2007). Epiblast SCs, in which Lif signaling was introduced transgenically (Bao et al., 2009), were shown to have regained the ability to participate in chimeric development and transmit to the germ-line, demonstrating that the loss of this signaling cascade during post-implantation development results, in part, with this diminishment of pluripotency.