Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses

Abstract

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.

Figure 1

Generation of mouse×nhpESC-GFP interspecies aggregation chimera. A: a ‘sandwich’ mouse aggregation chimera prepared by mixing a small clump of GFP-expressing nhp2706 ES cells (green; arrow) with two 2-cell mouse embryos in a depression well (arrowheads). B–F: confocal image of a fixed interspecies aggregation chimera (B: DIC; and C: DNA) produced with GFP-expressing BabESC-4, showing localization of BabESCs (D: green, arrowheads) at the mouse ICM [B,C,E: arrows; E: cdx-2, a trophectoderm specific marker, red] but not in the outer trophectodermal cells (F: merged imaged). Similar aggregation chimera w Bar=20 μm.

Figure 2

Developmental abnormalities in interspecies aggregation chimera at E10.5 prepared with GFP-expressing nhp 2706 ESCs. A–B: head–trunk axial deformity (A: BF; arrowheads, A: anterior head region; P: posterior trunk region) in an aggregation chimera. No GFP expression was observed (B: green). C–D: severely delayed embryonic development (C: BF), but no discernible GFP expression (D: green). The tail region was slightly damaged during dissection from the decidual sac. E–F: an implantation site without a definable embryo (E: BF) but with a few GFP expressing cells (F: green, arrowheads). BF: brightfield optics; A: anterior head region; P: posterior trunk region. Bars=500 μm.

Figure 3

Chimeric blastocyst injection embryos at E10.5 day post embryo transfer. A–B: Normal embryo produced from a mouse×GFP nhp2706-ESC injection chimera (A: BF), but without GFP expression (B: green). C–D: An abnormal mouse embryo (C: BF) derived from the transfer of a mouse×GFP-expressing nhp2706-ESCs injection chimeric. No GFP expressing cells are seen in the disorganized tissue (D: green, GFP). E–F: a reabsorbing mouse embryo (E: BF) derived from a mouse×GFP-expressing nhp2706-ESCs injection chimeric. A few GFP-expressing cells are observed in the fetus (F: green, arrowheads). G–H: Control chimeric embryo derived from a mouse×YFP-mouse ESCs blastocyst transfer into an ICR recipient. The normal embryo (I: BF) expresses YFP in many tissues (J: YFP). BF: bright field; GFP: green fluorescent protein. All embryo transfer were performed in ICR recipients. All chimera were produced with GFP-expressing rhesus 2706 male ESC line. Bar=500 μm.

Figure 4

PCR analysis of SRY (Left) and GFP (Right) DNA in embryos derived from mouse blastocyst injected with GFP nhp ESC 2706 male line. Lane 1: DNA marker; lane 2: chimeric outgrowth without GFP positive cell expression; lane 3: positive DNA control (transgenic monkey cells from ANDi); lanes 4–6: embryos derived from an injection chimera attempt (mouse×GFP nhpESC 2706 cell line); lane 8: DNA marker; lanes 9–13: embryos derived from an injection chimera (mouse×GFP nhpESC 2706 cell line); lane 14: positive DNA control (ANDi cells); lane 15: blank.

Figure 5

Interspecies chimeric outgrowth. A–C: Confocal imaging of a mouse blastocyst injected with male GFP-expressing nhp2706 ESCs at day 3 post-outgrowth. The GFP-nhpESCs remain clustered together (A: green) without intermixing with mouse ICM cells. B: Hoechst DNA; C: merged image. D–F: Sequential fluorescent and Hoffman Modulation Contrast (HMC) images of a mouse aggregation chimeric blastocyst produced with male GFP nhp2706 ESCs. At day 2 post outgrowth, the chimeric blastocyst attaches onto the MEF feeders with extensive GFP nhpESCs at the mouse ICM (D: green). On day 8 post-outgrowth (E), the expanding mouse ESCs (E: *) are distinctly separate from the expanding GFP-nhpESCs (E: green; inset, details of GFP-nhpESCs). On day 17 days post-outgrowth, rapid proliferation of the GFP-expressing nhpESCs along a distinct border of the largely differentiated mouse ESC colony (*) is observed, with little intermixing of the mouse: monkey ESCs. Mag=100×; Bar=20 μm.