GFP expressing Rhesus nhpESC lines Table

GFP-expressing Rhesus nhpESC lines (Table 1) used in the aggregation chimera assay produced expanded mouse blastocysts with GFP-expressing nhpESC lines 2706 and 3006 associated with the blastocyst inner cell mass (ICM) cells. We typically combined mouse zona pellucida-free 2-to-8-cell stage embryos with Rhesus or Baboon GFP-expressing nhpESCs (Rhesus: Fig. 1A, arrow) in a depression well and cultured them in vitro until the blastocyst stage (see Figs. S6–S8). After fixation, confocal optical sectioning (Baboon: Fig. 1B: differential interference contrast (DIC); 1C: Hoechst DNA, blue) demonstrated direct fluorescence detection of baboon GFP-expressing nhpESCs within the ICM (Fig. 1D: GFP, green, arrowheads). Here, no GFP-expressing baboon nhp 2706ESCs cells were observed in the outer trophectodermal cells, as demonstrated with a trophectoderm-specific antibody, CDX2 (Fig. 1E: red; arrow, ICM; composite image, Fig. 1F). We observed nearly 27% (129/479) of expanded chimera blastocysts with GFP-expressing Rhesus or Baboon nhpESCs exclusively in the mouse ICM and another 11% (55/479) with GFP-expressing Rhesus or Baboon nhpESCs in both the ICM and trophectoderm. The number of GFP-expressing nhpESCs associated with the mouse ICM was variable, with a majority showing 2–5 NHP cells within the ICM of expanded blastocysts. Time-lapse video microscopy (TLVM) was used to investigate intraspecific and interspecific chimera formation in vitro (Figs S6–S8). After 24–48h of aggregation within a depression well, compacted embryos with adhering mouse or Rhesus nhp ESCs were collected and prepared for TLVM recording for development to the expanded blastocyst stage. In intraspecific chimera (mouse×YFP-expressing mouse ESCs), video evidence suggested that the adhering mouse YFP-ESC (Fig. S6A–B) would integrate into the mouse during periods of rgd peptide expansion, often as trophectoderm cells were undergoing division. Interestingly, the expanding mouse blastocyst did not collapse during the YFP-ESC incorporation phase, perhaps suggesting that the mouse embryo and mouse ESCs share similar cell surface signaling molecules that could mediate aggregation. This observation was similar to control blastocysts lacking the zona pellucida (not shown), but distinct from observations reported in other rodents (Gonzales et al., 1996). We transferred mouse 2N embryo aggregation chimera produced with GFP-expressing nhp 2706 or 3006 ES cell lines into ICR strain recipients to investigate fetal contributions in vivo (Table 1). Of 57 total aggregation chimera transferred to 5 recipients, we observed 55 (96%; Table 1, column e) implantation sites but only 9 (16%; Table 1, column f) normal fetuses. The vast majority of the tissues recovered were either abnormal (25 total; 45%; Table 1, column g) or being reabsorbed (21 total; 38%; Table 1, column h). None of the fetuses recovered demonstrated GFP-expressing nhpESCs (Table 1, column i). Microscopic analysis of the fetal tissues recovered from embryos produced with rhesus GFP-expressing nhp2706 cell line showed many instances of axial abnormalities (head–trunk: Fig. 2A) and delayed fetal development (Fig. 2C) but no detectable GFP-expressing cells (Fig. 2B–D). In several instances, surviving GFP-expressing 2706 ES cells were observed in reabsorbing implantation tissues (Fig. 2E, brightfield; 2F, GFP, green, arrowheads). Thus, aggregation chimeras with GFP-expressing nhpESCs produced mosaic blastocysts with varying number of GFP cells associated with the mouse ICM and high numbers of abnormal fetuses following embryo transfer to pseudopregnant recipients. Next, we explored interspecies chimera after GFP-expressing nhp 2706 or 106 ESCs were injected into the expanded mouse blastocysts. We first determined the survival of injected nhp2706 ESCs within the mouse blastocoel niche. Chimeras were produced by microinjecting a known number of GFP-expressing nhp2706 ESCs into expanded mouse blastocysts (Fig. S2A), placing the GFP-expressing cells adjacent to the mouse ICM (Fig. S2B). As shown, within 4–6h, the re-expanded mouse blastocysts (Fig. S2C) demonstrated GFP-expressing nhp2706 ESCs in the blastocoel, some localized at the mouse ICM (Fig. S2D: GFP, green). However, only 43% [10/23] of injected blastocysts retained any GFP-positive nhp2706 ESCs after 21h of in vitro culture and fluorescent analysis of surviving GFP-nhp2706 ESCs revealed >70% loss of the total number of cells. We then performed embryo transfers of injected interspecies chimeric blastocysts to pseudopregnant ICR or NOD-SCID mice after using either rhesus or baboon GFP-expressing ESCs. We observed several implantation sites (ICR: 81/207 [39%]; NOD-SCID: 8/32 [25%]; Table 1, columns d–e) and a high percentage of normal E10.5 fetuses at recovery (ICR: 68/81 [84%]; NOD-SCID: 5/8 [63%]; also Fig. S3). However, none of the normal fetuses expressed GFP (Table 1; column i; Fig. S3). Conversely, from 48 embryo transfers using control intraspecific chimeric blastocysts produced with YFP-expressing mESCs, 16 implantation sites (33%; Table 1, column e) and 12 normal fetuses (75%; Table 1, column f) were recovered, with 5 fetuses expressing YFP (31%; Table 1, column i; see also, Fig. S3). Microscopic analysis of injection chimeric embryos produced with GFP-expressing Rhesus nhp 2706 ESCs (Fig. 3) or GFP-BabESC4 cell lines (Fig. S4) in either ICR or NOD-SCID recipients demonstrated no GFP expression in the tissues of recovered normal fetuses (Rhesus 2706: Fig. 3A–B; BabESC-4: Fig. S4A–B). Various abnormal embryos were largely negative for GFP detection also (Rhesus 2706: Fig. 3A–B), although occasional GFP ‘dots’ were observed in some recovered abnormal tissues (Rhesus 2706: Fig. 3E, brightfield; 3F, GFP, green, arrowheads; Fig. S4E, brightfield; Fig. S4F, GFP, green, arrowhead; BabESC-4: Fig. S4C, brightfield; S4D: GFP, green, arrowheads). Mouse intraspecific embryos produced with YFP-expressing mESCs and transferred to pseudopregnant recipients showed extensive fluorescence throughout the E10.5day fetus (Fig. 3G–H; YFP, yellow).