The long-term goal of this research is to establish transplantation of germ line stem cells (GSCs) as an approach to generate genetically modified large animal models of human and animal diseases, and to provide accessible systems to study the biology of GSCs in non-rodent animals. Introduction of genetic modification through the male germ line will shorten the time necessary to produce animal models and is potentially more efficient than the current method of somatic cell nuclear transfer. The testis stem cell is unique in that it is the only cell type in an adult individual that divides and contributes genes to subsequent generations, making it an ideal target for genetic modification. The pig is used as a non-rodent model because of its importance for biomedical research. Due to their size and physiology, results obtained in pigs are expected to be applicable to other large animal species and humans.
The aims of this renewal project are 1) to provide an efficient system for isolation and expansion of porcine GSCs;2) to generate pigs carrying a targeted gene deletion through germ cell transplantation;3) to study the effects of the testicular microenvironment on morphogenesis and germ cell differentiation from pluripotent stem cells using xenografting of isolated testis cells;and 4) to investigate the inductive effect of the microenvironment on transdifferentiation of porcine GSCs to somatic lineages. Experiments will explore enrichment and expansion of GSCs in stirred bioreactor suspension culture, transient stimulation of germ cell proliferation in co-culture with stable feeder lines expressing a VP22-c-Myc fusion protein, and application of recently developed transcription activator-like effector nuclease (TALEN) technology to GSCs for targeted gene modification. Generation of a pig model carrying a targeted mutation in the Duchenne's muscular dystrophy (DMD) gene through germ cell transplantation will provide proof-of- principle that the novel strategy developed in this project represents an efficient approach for the generation of loss-of-function large animal models. In addition, this pig model will provide a valuable resource for the study of DMD. De novo morphogenesis of functional testis tissue after grafting of isolated cells to mouse hosts was developed in the previous funding period. It allows reconstitution of spermatogenesis from isolated cells in an in vivo culture system, a process that is currently not possible in vitro. Thi system will serve as a bioassay for germ lineage differentiation from porcine induced pluripotent stem cells, and will enable us to examine the functional role of primary cilia on testicular somati cells during testicular morphogenesis and establishment of the stem cell niche. Finally, the project will employ unique in vitro and in vivo assays to explore direct differentiation of GSCs into epithelial or vascular endothelial cells following exposure to an inductive microenvironment. Overall, the complementary strategies of homologous germ cell transplantation and testis cell grafting to be employed in this proposal will enable efficient germ line gene deletion in a large animal model and will establish a pre-clinical platform for work with GSCs in reproductive and regenerative medicine.

Public Health Relevance

To study and design treatments for human and animal diseases requires appropriate animal models. While a large number of rodent models exist, it is increasingly recognized that non-rodent animal models like pigs and non-human primates more closely resemble human physiology and size. This project will provide a novel strategy to generate non-rodent animal models carrying a targeted gene mutation that is potentially faster and more efficient than the currently used approach of cloning. In addition, it will allow investigation of germ line stem cell biology in non-rodent species and may lead to new approaches to use stem cells in regenerative medicine.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Research Project (R01)
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Special Emphasis Panel (ZTR1-CM-6 (01))
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Mirochnitchenko, Oleg
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University of Calgary
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T2 1-N4
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
Ou, Young; Dores, Camila; Rodriguez-Sosa, Jose-Rafael et al. (2014) Primary cilia in the developing pig testis. Cell Tissue Res 358:597-605
Dores, Camila; Dobrinski, Ina (2014) De novo morphogenesis of testis tissue: an improved bioassay to investigate the role of VEGF165 during testis formation. Reproduction 148:109-17
Arregui, LucĂ­a; Dobrinski, Ina (2014) Xenografting of testicular tissue pieces: 12 years of an in vivo spermatogenesis system. Reproduction 148:R71-84