Induced pluripotent cells as an alternative to produce viable gametes

• Ano de defesa: 2025
• Autor(a) principal: Recchia, Kaiana
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: In vitro gametogenesis; In vitro myogenesis; Bovine; Bovino; Gametogênese in vitro; iPSCs; Miogênese in vitro; Pluripotência; Pluripotency
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/10/10132/tde-06022026-112644/

Bovine is a highly relevant model for biotechnology and animal production, being strategic for translational studies and the development of new technologies, such as cellular agriculture and assisted reproduction. Bovine induced pluripotent stem cells (biPSCs) offer a powerful tool to investigate cell differentiation, the production of cultured meat, and in vitrogametogenesis, enabling advances in both practical applications and experimental models for genetic improvement, the aim of this thesis is to generate biPSCs using both non-integrative and integrative approaches and to evaluate their plasticity through in vitro gametogenesis. In Chapter 2, non-invasive cell collection and isolation are tested, followed by their reprogramming and differentiation into the germline lineage. Cells derived from urine (bUDCs) and milk (bMDCs) were successfully isolated, with the use of UDCs representing a novel, non- invasive approach in the bovine model. The reprogramming of bUDC1 into biPSCs, although dependent on exogenous mOSKM factors, was successfully induced into bovine primordial germ celllike cells (bPGCLCs), which were cultured in the xrOvary system. Cyst-like structures similar to those reported in mice were observed, representing the first description of this system in bovines. In Chapter 3, the objective was to develop a non-integrative protocol for the bovine model and to assess whether the generated biPSCs can contribute to muscle lineages. In this chapter it is reported for the first successful episomal reprogramming of bovine fetal fibroblasts (bFFs) using the pMasterK and pMaster12 plasmids. Differentiation of episomal biPSCs into myogenic lineages resulted in multinucleated cells expressing bMYOG transcripts, typical of myotubes. This is a novel finding in bovines, with potential applications for cultured meat production from biPSCs. In chapter 4, the objective was to generate biPSCs using different reprogramming methodologies and to isolate bESCs (bovine embryonic stem cells) and differentiate them into bPGCLCs using different protocols. Multiple biPSC lines derived from bFFs were established using different reprogramming methodologies, including the unprecedented reprogramming of bovine UDCs, and a bESC line was also derived. biPSCs were induced into bPGCLCs using three pre-induction protocols. Spheroid formation was observed in protocols 2 and 3, and transcripts related to germline lineages and epigenetic regulators (bPOU5F1, bTFAP2C, bPRDM1, bSOX17, bDDX4, bPRDM14, bDNMT1, and bDNMT3a) were analyzed. The resulting bPGCLCs demonstrate potential for in vitro gametogenesis, enabling the generation of functional gametes, accelerating genetic improvement, and contributing to breed conservation. Overall, the findings reported here confirm the plasticity of biPSCs obtained via non-integrative and integrative protocols and the successful establishment of differentiation protocols. These results are unprecedented and highlight biPSCs as a key tool in animal biotechnology and translational applications, aligned with global trends in research and innovation.