Interspecies Chimerism with Mammalian Pluripotent Stem Cells

Abstract

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Keywords: CRISPR-Cas9; human naïve pluripotent stem cells; human-cattle chimeric embryo; human-pig chimeric embryo; interspecies blastocyst complementation; interspecies chimera; organ and tissue generation; pluripotent stem cells; zygote genome editing.

Figure 1. Interspecies Rat-Mouse Chimeras Derived from Rat PSCs

(A) Rat-mouse chimeras generated by rat ESCs (DAC2). Left, an E18.5 rat-mouse chimeric fetus. Red, hKO-labeled rat cells. Right, a 12-month-old (top) and 24-month-old (bottom) rat-mouse chimera. (B) Chimera forming efficiencies with rat ESC lines (DAC2 and DAC8) and rat iPSC lines (SDFE and SDFF). n, number of embryo transfers. (C) Representative fluorescence images showing hKO-labeled rat ESCs (DAC2) contributed to different tissues in the 24-month-old rat-mouse chimera. Red, hKO-labeled rat cells. Blue, DAPI. Scale bar, 100 μm. (D) Representative immunofluorescence images showing the expression of aging-related histone marks, including H3K9me3 and H4K20me3, in the kidney tissue of neonatal and 24-month-old chimeras. Scale bar, 10 μm. (E) Levels of chimerism of rat ESCs (DAC2) in different tissues of the 24-month-old rat-mouse chimera. Error bars indicate SD. (F) Rat iPSCs (SDFE) contributed to the neonatal mouse gall bladder. Left, bright-field (top) and fluorescence (bottom) images showing a neonatal mouse gallbladder contained cells derived from rat iPSCs. White arrowheads indicate the gallbladder. Right, representative immunofluorescence images showing the expression of a gallbladder epithelium marker (EpCAM) by rat cells. Red, hKO-labeled rat cells; blue, DAPI. Scale bar, 50 μm. See also Figure S1 and Table S2.

Figure 2. Interspecies Blastocyst Complementation via CRISPR-Cas9-Mediated Zygote Genome Editing

(A) Schematic of the CRISPR-Cas9 mediated rat-mouse blastocyst complementation strategy. (B) Left, bright-field (top) and fluorescence (bottom) images showing the enrichment of rat cells in the pancreas of an E18.5 Pdx1^−/− mouse. Li, liver; St, stomach; Sp, spleen. Yellow-dotted line encircles the pancreas. Red, hKO-labeled rat cells. Middle and right (top), representative immunofluorescence images showing rat cells expressed α-amylase in the Pdx1^−/− mouse pancreas. Blue, DAPI. Right (bottom), a representative immunofluorescence image showing that some pancreatic endothelial cells, as marked by a CD31 antibody, were not derived from rat PSCs. Scale bar, 100 μm. (C) Bright field (left) and fluorescence (right) images showing the enrichment of rat cells in the heart of an E10.5 Nkx2.5^−/− mouse. Red, hKO-labeled rat cells. (D) Bright field (top) and fluorescence (bottom) images showing the enrichment of rat cells in the eye of a neonatal Pax6^−/− mouse. Red, hKO-labeled rat cells. WT, mouse control; WT+rPSCs, control rat-mouse chimera without Cas9/sgRNA injection. See also Figure S2 and Tables S1 and S2.

Figure 3. Naive Rodent PSCs Fail to Contribute to Chimera Formation in Pigs

(A) Schematic of the generation and analyses of post-implantation pig embryos derived from blastocyst injection of naive rodent PSCs. (B) Summary of the pig embryos recovered between day 21–28 of pregnancy. (C) Genomic PCR analyses of pig embryos derived from blastocyst injection of mouse iPSCs or rat ESCs. Mouse- and rat- specific mtDNA primers were used for the detection of chimeric contribution from mouse iPSCs and rat ESCs, respectively. Pig-specific mtDNA primers were used for the control. See also Tables S2 and S3.

Figure 4. Generation and Interspecies ICM Incorporation of Different Types of hiPSCs

(A) Schematic of the strategy for generating naive, intermediate, and primed hiPSCs. (B) (Top) Representative bright-field images showing the colony morphologies of naive (2iLD-, 4i-, and NHSM-hiPSCs) and intermediate (FAC-hiPSCs) hiPSCs. Bottom, representative immunofluorescence images of naive and intermediate hiPSCs stained with an anti-OCT4 antibody. Red, OCT4; blue, DAPI. Scale bar, 100 μm. (C) Schematic of the experimental procedures for producing cattle and pig blastocysts obtained from in vitro fertilization (IVF) and parthenoactivation, respectively. Blastocysts were subsequently used for laser-assisted blastocyst injection of hiPSCs. After hiPSC injection, blastocysts were cultured in vitro for 2 days before fixation and analyzed by immunostaining with an anti-HuNu and an anti-SOX2 antibodies. Criteria to evaluate the survival of human cells, as well as the degree and efficiency of ICM incorporation are shown in the blue box. (D) Number of hiPSCs that integrated into the cattle (left) and pig (right) ICMs after ten hiPSCs were injected into the blastocyst followed by 2 days of in vitro culture. Red line, the average number of ICM-incorporated hiPSCs. Blue dot, the number of ICM-incorporated hiPSCs in each blastocyst. See also Figure S3 and Table S4.

Figure 5. Generation of Post-implantation Human-Pig Chimeric Embryos

(A) Schematic of the experimental procedures for the generation and analyses of post-implantation pig embryos derived from blastocyst injection of naive and intermediate hiPSCs. (B) Summary of the pig embryos recovered between day 21–28 of pregnancy. (C) Bar graph showing proportions of normal size and growth retarded embryos, as well as the proportion of fluorescence-positive and -negative embryos, generated from different types of hiPSCs. (D) Bar graph showing the proportion of normal size and growth-retarded embryos (among those exhibiting a fluorescence signal) generated from different types of hiPSCs. (E) Bar graph showing the proportion of normal-sized and growth-retarded embryos (among those without exhibiting a fluorescence signal) generated from different types of hiPSCs. See also Figure S4 and Tables S5 and S6.

Figure 6. Chimeric Contribution of hiPSCs to Post-implantation Pig Embryos

(A) Representative bright field (left top) fluorescence (left bottom and middle) and immunofluorescence (right) images of GFP-labeled FAC-hiPSCs derivatives in a normal size day 28 pig embryo (FAC #1). Scale bar, 100 μm. (B) Representative immunofluorescence images showing chimeric contribution and differentiation of FAC-hiPSCs in a normal size, day 28 pig embryo (FAC #1). FAC-hiPSC derivatives are visualized by antibodies against GFP (top), TUJ1, SMA, CK8 and HuNu (middle). (Bottom) Merged images with DAPI. Insets are higher magnification images of boxed regions. Scale bar, 100 μm. (C) Representative gel images showing genomic PCR analyses of pig embryos derived from blastocyst injection of 2iLD-iPSCs (surrogates #8164 and #20749) and FAC-hiPSCs (surrogates #9159 and #18771) using a human specific Alu primer. A pig specific primer Cyt b was used for loading control. nc, negative control with no genomic DNA loaded. pc, positive controls with human cells. Pig 1D, 1G, and 1I, pig controls. ID, surrogate and pig embryos. See also Figure S5 and Table S2.