Parthenogenetic Stem Cells from Unfertilized Porcine Oocytes

Mammalian parthenogenesis remains significantly understudied due to fundamental biological limitations arising from genomic imprinting, where the absence of paternally imprinted genes leads to developmental abnormalities and embryonic arrest. While parthenogenetic stem cells have been derived from mouse and human sources, extending this research to translationally relevant large animal models like pigs represents a critical knowledge gap, despite their physiological and anatomical similarities to humans making them ideal for regenerative medicine applications. This thesis aimed to establish efficient protocols for generating and characterising parthenogenetic stem cells from unfertilised porcine oocytes, focusing on optimising activation methods, culture conditions, and comprehensive characterisation of the derived stem cells. Additionally, it aimed to develop protocols for the cardiac differentiation of porcine parthenogenetic stem cells to establish the basis for subsequent applications in regenerative medicine. In this thesis, novel porcine parthenogenetic stem cell lines (pPSCs) were generated (n=24 from n=2,578 oocytes) and characterized, which included a comparison to porcine embryonic stem cell (pESCs) lines (n=10 from n=2,917 in vitro fertilized oocytes) and induced pluripotent stem cell (piPSC) lines (n=1). Molecular and immunofluorescence characterisation confirmed authentic pluripotent markers, including OCT4A, NANOG, and SALL4, while maintaining normal diploid karyotypes (2n = 38) across all examined cell lines (n=5 pPSC lines, n=5 pESC lines, and n=1 piPSC line). Bulk RNA sequencing analysis revealed distinct transcriptional profiles in the tested pluripotent stem cell lines, demonstrating particularly strong signatures of naïve pluripotency in pPSCs. Finally, first evidence for cardiac differentiation of porcine parthenogenetic stem cells is presented, demonstrating robust expression of cardiac-specific markers (cTnT, α-actinin) with characteristic cross-striated patterns and spontaneous contractile activity. The use of pPSC derivatives offers clear advantages in regenerative medicine applications, because of their largely homozygous genome, including haploidentity of the coding sequences for the immunologically highly relevant multi-histocompatibility complex (MHC) proteins. This trait offers opportunities for "off-the-shelf” therapeutic use with minimal immunosuppression or no immune suppression, the latter being a major limitation in classical allograft approaches. The pPSC model is considered a stepping stone to enable pivotal preclinical stepping stones on the way to clinical translation.