Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8‐cell embryo injection and aggregation

Abstract

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8‐cell embryo aggregation, 8‐cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8‐cell embryo injection and 8‐cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8‐cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte‐derived drug‐metabolizing enzyme. Collectively, these findings indicate that the 8‐cell embryo injection method is the most suitable for generation of PSC‐derived organs via chimeric animal generation, particularly for the liver.

Generation of Lungs by Blastocyst Complementation in Apneumic Fgf10-Deficient Mice

“…Given the low efficiency of generating lung organs by intraspecies blastocyst complementation (Table 1), whether PSCderived lung organs could be generated in Fgf10-knockout rodents or livestock by interspecies blastocyst complementation remains to be investigated in a subsequent study. Efficient chimerism is considered to be the most important factor when interspecies chimeras are produced for organ generation (Okumura et al, 2019). Mouse PSCs contribute to kidney generation in rats (Goto et al, 2019), whereas rat PSCs failed to generate kidneys in anephric Sall1 mutant mice due to their poor contribution to the metanephric mesenchyme (Usui et al, 2012).…”

“…Furthermore, although human PSCs contribute to pig embryos by blastocyst injection, the level of chimerism was low and insufficient for organ generation (Wu et al, 2017). Eight-cell embryo injection increased the contribution rate of rat ESCs to chimeric mice but decreased the chimera generation rate, compared with blastocyst injection (Okumura et al, 2019). The pluripotent state affects the contribution of PSCs to xenogeneic embryos; thus, matching the developmental timing may be critical for successful chimera formation (Wu et al, 2017;Huang et al, 2018).…”

Generation of donor organs in chimeric animals via blastocyst complementation

“…In their study Okumura and colleagues compared the degree of distribution of rat cells in chimeric rat-mouse embryos by different methods: the 8-cell aggregation method, injection into an 8-cell embryo, and injection into a blastocyst. According to the study, the degree of chimerism was highest when researchers used the injection method into an 8-cell embryo, although the percentage of chimeric mice was higher when they injected cells into the blastocyst (Okumura et al, 2019).…”

Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection

“…have reported a success rate of 29% in obtaining chimeric pups by microinjections into blastocysts followed by uterus transfer, with this method resulting in low degrees of chimerism in some organs (particularly liver). Although we did not do the uterus transfers into mouse fosters, the uterus transfer into rats using our protocol had a success rate of 36% in obtaining chimeric pups, and the chimerism in all tissues analyzed, including brain (the tissue not assessed by Okumura et al., 2019 ) and liver, was readily detectable by standard PCR in all the chimeras obtained using our protocol.…”

“…A recently published protocol for generating rat–>mouse chimeras compared three different methods: (1) 8-cell stage embryo aggregation (co-culture of ES cells and embryo); (2) ES cell microinjection into 8-cell stage embryos and; (3) ES cell microinjection into blastocysts ( Okumura et al., 2019 ). The microinjected 8-cell stage embryos in that study were incubated in vitro until the blastocyst stage and then transferred into the mouse uteri.…”