Generation of functional xenogeneic germ and muscle stem cells via interspecies chimerism
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Author
Date
2023Type
- Doctoral Thesis
ETH Bibliography
yes
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Abstract
Pluripotent stem cells (PSCs), including embryonic and induced pluripotent stem cells (ESCs and iPSCs) can contribute to all germ layers of the developing embryo when injected into host blastocysts, which results in chimeric animals. These properties can be harnessed to generate donor PSC derived cells and tissues across species boundaries. Blastocyst complementation is a promising technique exploiting this observation, wherein the host blastocysts carry mutations hindering the formation of specific cells, allowing the injected PSCs to populate this developmental niche and generate cells or tissues exclusively derived from the donor PSCs.
Generation of male gametes in vitro is investigated not only for the study of how germ cells are formed, but also for potential species conservation efforts. However, it often relies on testes of the species of interest being available, which is not feasible for rare species. To overcome this limitation, in the first study, we aimed to generate exclusively donor PSC derived spermatozoa via blastocyst complementation with mouse-to-mouse intra- and rat-to-mouse interspecies chimeras. To this end, we injected the donor cells into mouse Tsc22d3-knockout (KO) blastocysts, which carried a defect in meiosis I, causing male sterility in non-complemented animals. Resultant intraspecies chimeras harbored exclusively allogeneic donor mouse PSC derived spermatozoa, and were fertile upon natural mating with female mice. In addition, injection of rat rESCs into Tsc22d3-KO blastocysts resulted in mouse-rat chimeras, and analysis of testes and spermatozoa revealed exclusive generation of xenogeneic donor rESC derived germ cells, which was further confirmed by single-cell RNA-sequencing. Fertilizing rat oocytes using chimera derived rat spermatozoa led to the generation of blastocysts in vitro, and implantation sites and embryos in vivo, although no offspring was generated.
In the second project, blastocyst injections were utilized to generate muscle stem cells, also termed satellite cells, conferring the skeletal muscle’s immense capacity for regeneration, which is lost in muscular dystrophies. We generated iPSCs from a muscular dystrophy mouse model, gene-corrected the present mutation using CRISPR/Cas9 and utilized the iPSCs to generate intraspecies chimeras. The host blastocysts carried a satellite cell specific ablation system allowing for exclusive generation of donor cell derived satellite cells. Upon isolation of the iPSC derived satellite cells, and in vitro propagation, the resulting myoblasts were transplanted into the Tibialis anterior muscles of dystrophic mice, where they expressed dystrophin. Further, we generated interspecies chimeras via injection of the iPSCs into rat blastocysts. We were able to purify mouse satellite cells from these chimeras, expanded and characterized them in vitro, and we observed contribution of the myoblasts to dystrophin expression in vivo in dystrophic recipient muscles.
These results show that generation of interspecies chimeras carries the potential to not only generate xenogeneic germ cells, potentially capable of aiding in species conservation, but also adult muscle stem cells, which could help in paving the way to treat muscular dystrophies in human patients. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000603822Publication status
publishedExternal links
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Contributors
Examiner: Bar-Nur, Ori
Examiner: Drukker, Micha
Examiner: Tedesco, Francesco
Examiner: von Meyenn, Ferdinand
Publisher
ETH ZurichSubject
Stem cells; Stem cell-based therapeutic approaches; Male germ cells; Interspecies ChimerismOrganisational unit
09626 - Bar-Nur, Ori / Bar-Nur, Ori
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