In vivo and in vitro developmental study of porcine testis cells and tissue
Date
2021-12-16
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-0306-497X
Type
Thesis
Degree Level
Doctoral
Abstract
Testis development and function involve multiple cell types and require complex cellular interactions and signaling cascades; hence, the study of these processes necessitates improvement of ex situ models of testicular cells or tissue. Accordingly, objectives of the first and second studies in this thesis were to improve in vitro culture of testis cells by testing the effects of various culture components on proliferation, colony-formation, and potency of germline stem cells. The examined components included different media, serum types, and several key growth factors, tested in sequential experiments using a factorial design. The third study focused on assessment of pluripotency of cultured neonatal porcine gonocytes and their potential to trans-differentiae into somatic cells of different cell lineages. The goal of our fourth study was to investigate the feasibility of applying ultrasound biomicroscopy (UBM) for the assessment of testis tissue grafts and cell implants by initially validating its use in a short-term study. In the fifth study, we examined repeated application of UBM in a longitudinal manner to test its reliability and accuracy in representing the developmental changes in grafts and implants. As a result, we showed increased proliferation of porcine gonocytes with a given culture condition combination (DMEM+15% FBS), followed by attainment of pluripotency markers by cultured gonocytes and their colonies, indicative of their spontaneous transformation into a pluripotent state. Supplementation of certain growth factors at a given concentration to the base media also led to further increases in gonocyte numbers and colony diameters, while maintaining their expression of pluripotency and germ cell markers. We also observed successful derivation of somatic cells of different lineages from germ cell originated-pluripotent stem cells. Furthermore, application of UBM for non-invasive in vivo developmental study of testicular grafts and implants was shown to be accurate and reliable, thereby reducing the number of animals needed in future similar experiments. Taken together, the studies describe improved germ cell culture conditions, transformation of gonocytes into a pluripotent state, as well as validating and using UBM as an accurate and reliable tool for in vivo developmental study of testis grafts and implants.
Description
Keywords
Germline stem cells, Gonocytes, Culture, Differentiation, Trans-differentiation, Pluripotency, Testicular development, Testis tissue xenografting, Testis cell aggregate implantation, Ultrasound biomicroscopy
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Biomedical Sciences
Program
Veterinary Biomedical Sciences