ESC pluripotency has most convincingly been demonstrated in

ESC pluripotency has most convincingly been demonstrated in reaggregated embryos where the resultant offspring have ESC contributions to all germ layers and tissues, including the germ line (reviewed by Rossant, 2001). Thus far, only mouse and rat embryonic stem cells (mESCs) aggregated with mouse or rat embryos result in offspring born with demonstrated ESC contribution to all three germ layers and the germ line (Iannaccone and Jacob, 2009). Currently, hESC differentiation is assayed by embryoid bodies (EBs) or teratomas and both contribute to all three germ layers (Conley et al., 2005), but these technologies have limitations—EBs do not mimic 3D axial morphogenesis in vitro accurately and teratomas are a foreign environment that do not produce germ cells. Overwhelming ethical concerns obviously preclude interspecific chimera attempts with hESCs. However, the derivation of nonhuman primate ESCs (nhpESCs) can responsibly bridge gaps in our scientific knowledge between mESCs and hESCs, for example, in the generation of chimeric nonhuman primates with nhpESCs cells, although issues with NHP embryo availability, cost, and complex technical obstacles with chimera production remain (Takada et al., 2002; Schramm and Paprocki, 2004; Scott, 2006; Roberts, 2005). Thus, Mitalipov et al. (Mitalipov et al., 2006) had previously demonstrated that while derived GFP-expressing rhesus pluripotent ESCs injected into 4-to-8-stage fertilized rhesus embryos would incorporate into the trophectoderm and ICM cells of the expanded purchase LY 2109761 grown in vitro, efforts to produce a chimeric monkey after embryo transfer did not succeed. Interspecies chimeras have been advanced as an alternative process for exploring early human developmental processes and helping address the basic embryology of hESCs and their potential applications in cell-based therapies (James et al., 2006). The mouse is the best-characterized mammalian model and perhaps a logical choice for studying interspecies chimera: there is an abundance of cheap embryos and recipients; an enormous background literature on mESCs already exists; and mouse–mouse chimeras are well established with regards to genetics, strains and proven techniques. Furthermore, mESCs are unencumbered by the material transfer agreement (MTA) restrictions currently imposed on all NIH Registry hESC lines that explicitly prohibit their use in animal chimera production. Taken together, the answers obtained from testing interspecies mouse–NHP chimeras in utero, after chimeric embryo transfer, and in vitro might provide new and important information on the developmental potentials of embryonic stem cells. In addition, if successful, these intraspecific chimera would open innovative methods for preserving germ lines from endangered species (Songsasen and Wildt, 2007; Pukazhenthi et al., 2006) as well as specialty biomedical research models (Yang et al., 2008; Chan and Yang, 2009). Here, we explore mouse–nhpESC chimeras produced with classic mouse embryo aggregation or blastocyst injection techniques (Nagy, 2003). Using GFP-expressing rhesus and baboon nhpESCs, we demonstrate that nhpESCs associate with the ICM in expanded mouse blastocysts, but rarely proliferate after outgrowth experiments and do not intermingle with mouse tissues, as determined by in vitro analysis. Furthermore, we show that chimeric mouse–nhpESC blastocysts transferred to pseudopregnant mouse recipients produce fetuses but without detectable contribution from the GFP-expressing nhpESCs, as ascertained by immunohistochemical, PCR and MRI analysis. Collectively, we conclude that interspecies chimera between distant mammals is unfavorable for studying the full pluripotency of primate ESCs, lending intellectual support for intraspecific primate chimeric experimentation.
Results The rhesus male line nhpESC 2706 was particularly robust following transduction with the EF1α-GFP transgene and could be traced in mouse chimera tissues using monkey-specific primers to the SRY gene. Supplemental Table S1 summarizes the various stem cell lines employed for preparing injection- or aggregation-produced interspecies mouse chimeras. All of the nhpESCs employed were low passage colonies (range: 7–51) of good ESC morphology (Fig. S1A) and maintained their pluripotent characteristics following transduction with various GFP transgenes (Fig. S1B), as determined by ‘stemness’ (Fig. S1C; Table S1) and cell surface marker expression (Table S1), as well as their ability to produce teratomas when injected into NOD-SCID strain mice. Additionally, spontaneous differentiation of GFP-expressing nhpESC colonies in vitro did not silence the transgene, providing confidence that the primate cells would maintain GFP detection within interspecific chimera construction following embryo transfer (Fig S1D–I). Control intraspecific chimeras were produced by a yellow fluorescent variant of R1 mESCs (7ACS/EYFP; ATCC; Manassas, VA) that was germline-competent and stained positive for pluripotency markers (Hadjantonakis et al., 2002).