The long-term goals of this project are to establish germline stem cell (GSC)-based gene editing to generate large animal models of human diseases and for regenerative medicine (PAR-16-093), and to provide accessible systems to study the GSC niche in non-rodent animals. The testis stem cell is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Pigs are important models for pre-clinical research because they are phylogenetically and physiologically more similar to humans than rodents. Gene editing through GSCs rather than embryos will shorten the time necessary to produce germline gene edited animal models. It is also more broadly applicable to disease models where gene dosage and epigenetics have a role and smaller strains of pigs are required.
The aims of this renewal project are 1) to establish a culture system that promotes porcine germ cell expansion in vitro; 2) to explore the role of testicular somatic cells in formation of a functional GSC niche; and 3) to develop a precise approach for targeted gene editing in porcine GSCs. While germline transmission of a genetic modification is possible with transplantation of primary pGSCs, efficiency of the approach would benefit from a robust in vitro system for expansion of edited cells prior to transplantation. To accomplish this goal, we will define the metabolic phenotype of pGSCs to inform design of appropriate culture conditions. Grafting to mouse hosts and transplantation to recipient pigs will monitor the ability of cultured cells to support spermatogenesis as functional endpoint. Germ cell function is controlled by interaction with the niche microenvironment. Our novel testicular organoid system and xenografting of testis cells will enable introduction of cell type-specific modifications and characterize stem cell-niche interactions. We will test the hypothesis that primary cilia on testicular somatic cells are essential for niche formation, and that the somatic environment modulates germ cell differentiation and supports germ lineage differentiation of porcine induced pluripotent stem cells (piPSCs). Finally, we will test the hypothesis that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated homology directed repair (HDR) will allow efficient targeted genome editing in GSCs for precise replication of human disease alleles. As HDR is limited to small DNA alterations (~1-50 bp), we will also explore Precise Integration into Target Chromosome (PITCh) for targeted gene editing to enable introduction of large transgenes and/or humanizing parts of the pig genome for regenerative medicine. Introduction of a mutant porcine insulin gene (INSC94Y) that matches the human INSC96Y mutation present in permanent neonatal diabetes mellitus will provide proof-of-principle that our novel strategies are feasible and efficient. Overall, this work will establish precise targeted germline gene editing in pigs, and further define stem cell-niche interactions.

Public Health Relevance

To study and design treatments for human diseases requires appropriate animal models. Despite the availability of many rodent models, findings in rodents do not readily translate to human patients but large animal models, such as pigs and non-human primates, closely resemble human physiology and size. This project will provide a novel strategy for precise genetic modification in testis stem cells to generate large animal models of human diseases and for regenerative medicine, and will establish accessible systems to study germline stem cell biology in non-rodent animals.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Mirochnitchenko, Oleg
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University of Calgary
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T2 1N4
Tang, Lin; Bondareva, Alla; González, Raquel et al. (2018) TALEN-mediated gene targeting in porcine spermatogonia. Mol Reprod Dev 85:250-261
Valenzuela-Leon, Paula; Dobrinski, Ina (2017) Exposure to phthalate esters induces an autophagic response in male germ cells. Environ Epigenet 3:dvx010
Zeng, W; Alpaugh, W; Stefanovski, D et al. (2017) Xenografting of isolated equine (Equus caballus) testis cells results in de novo morphogenesis of seminiferous tubules but not spermatogenesis. Andrology 5:336-346
Dores, Camila; Alpaugh, Whitney; Su, Lin et al. (2017) Primary cilia on porcine testicular somatic cells and their role in hedgehog signaling and tubular morphogenesis in vitro. Cell Tissue Res 368:215-223
Sakib, Sadman; Dores, Camila; Rancourt, Derrick et al. (2016) Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment. Methods Mol Biol 1502:111-8
Tang, L; González, R; Dobrinski, I (2015) Germline modification of domestic animals. Anim Reprod 12:93-104
González, Raquel; Dobrinski, Ina (2015) Beyond the mouse monopoly: studying the male germ line in domestic animal models. ILAR J 56:83-98
Dores, C; Rancourt, D; Dobrinski, I (2015) Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis. Andrology 3:590-7
Ou, Young; Dores, Camila; Rodriguez-Sosa, Jose-Rafael et al. (2014) Primary cilia in the developing pig testis. Cell Tissue Res 358:597-605
Rodriguez-Sosa, Jose R; Bondareva, Alla; Tang, Lin et al. (2014) Phthalate esters affect maturation and function of primate testis tissue ectopically grafted in mice. Mol Cell Endocrinol 398:89-100

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