In vitro cellular reprogramming into pluripotency (generation of iPS cells) in the porcine model using non-transgenic and non-invasive methodology

Author(s)

Kaiana Recchia

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2020-12-17

Examining board members

Fabiana Fernandes Bressan; Moyses dos Santos Miranda; Kelly Cristine Santos Roballo

• Advisor: Fabiana Fernandes Bressan

Abstract

The swine is an attractive biomedical model because it shares immunological, anatomical, and physiological similarities with the human. It is advantageous to reprogram cells into pluripotency in this model to develop translational studies in human and veterinary regenerative medicine. In addition, iPSCs derived from cells collected in a non-invasive or less invasive methodology could have their use simplified, and is unprecedented in this animal model. Hence, this study aims to produce swine iPSCs cells in vitro from cells derived from urine (urine-derived cells or UDCs) and peripheral blood mononuclear cells (PBMCs) by non-invasive or less invasive collection, respectively. Therefore, three swine females had their urine and peripheral blood collected. The collected blood was processed by density gradient separation, and erythroblast expansion was performed; however, the number of cells necessary for reprogramming was not obtained in our conditions. From the processed urine, we observed UDC cells after approximately one week in culture. One of the isolated lineages (UDC3) was subjected to the reprogramming processes, either using an integrative methodology through lentiviral transduction with the transcription factors OCT4, SOX2, KLF4, and C-MYC (OSKM, human or murine) or using a non-integrative methodology with episomal vectors containing the C-MYC, LIN28, SOX2, KLF4, OCT3 / 4 and shp53 factors. However, only colonies derived from integrative reprogramming were obtained, from which three clonal lineages (C1, C2, and C3) were isolated and maintained in culture for at least 28 passages, demonstrating the ability of self-renewal in vitro. The lineages were characterized for their morphology, the detection of proteins related to pluripotency by immunofluorescence, the quantification of endogenous and exogenous transcripts, and the ability to form embryoid bodies. The swine in vitro reprogrammed cells obtained in this study were obtained from UDCs and characterized. They may enable the future development of new technologies applied using the swine model, benefiting both animal production and translational science, aiming its use as a biomedical model in vivo. (AU)

• FAPESP's process: 19/02811-2 - In vitro cellular reprogramming to pluripotency (iPS) in the swine model from non-transgenic and non-invasive methodology
• Grantee: Kaiana Recchia
• Support Opportunities: Scholarships in Brazil - Master