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©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
In vitro induced pluripotency from urine-derived cells in porcine
Kaiana Recchia, Lucas Simões Machado, Ramon Cesar Botigelli, Naira Caroline Godoy Pieri, Gabriela Barbosa, Raquel Vasconcelos Guimarães de Castro, Mariana Groke Marques, Laís Vicari de Figueiredo Pessôa, Paulo Fantinato Neto, Flávio Vieira Meirelles, Aline Fernanda de Souza, Simone Maria Massami Kitamura Martins, Fabiana Fernandes Bressan
Kaiana Recchia, Lucas Simões Machado, Flávio Vieira Meirelles, Fabiana Fernandes Bressan, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
Ramon Cesar Botigelli, Department of Pharmacology and Biotechnology, Institute of Bioscience, São Paulo State University, Botucatu 18618-689, São Paulo, Brazil
Naira Caroline Godoy Pieri, Gabriela Barbosa, Raquel Vasconcelos Guimarães de Castro, Laís Vicari de Figueiredo Pessôa, Paulo Fantinato Neto, Flávio Vieira Meirelles, Aline Fernanda de Souza, Fabiana Fernandes Bressan, Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
Mariana Groke Marques, Embrapa Suínos e Aves, Empresa Brasileira de Pesquisa Agropecuária, Concordia 89715-899, Santa Catarina, Brazil
Simone Maria Massami Kitamura Martins, Department of Animal Sciences, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
Author contributions: Recchia K and Bressan FF conceptualized the experiments and wrote the manuscript; Recchia K, Machado LS, Barbosa G, Marques MG and Martins SMMK collected and processed samples; Recchia K, Botigelli RC, Pieri NCG, de Castro RVG, Meirelles FV, de Souza AF, Pessôa LVF, Fantinato Neto P and Bressan FF collected data and interpreted the collected data; all authors revised the manuscript.
Supported by the São Paulo Research Foundation (FAPESP) - Brazil (financial support grants #2015/26818-5, #2013/08135-2, #2019/02811-2); Coordination of Superior Level Staff Improvement (CAPES 23038.006964/2014-43 and financial code 001) and National Council for Scientific and Technological Development (CNPq 433133/2018-0).
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo.
Institutional animal care and use committee statement: All procedures were performed following the National Council for Control of Animal Experimentation (CONCEA) rules and were approved by the Ethics Committee on Animal Experimentation of the Faculty of Animal Science and Food Engineering and Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo (protocols 6372070119 and 7051150717).
Conflict-of-interest statement: The authors declare that there are no conflicts of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Fabiana Fernandes Bressan, DVM, Professor, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av Duque de Caxias Norte 225, Pirassununga 13635-000, São Paulo, Brazil.
fabianabressan@usp.br
Received: August 23, 2021
Peer-review started: August 23, 2021
First decision: October 3, 2021
Revised: October 11, 2021
Accepted: February 15, 2022
Article in press: February 15, 2022
Published online: March 26, 2022
Processing time: 213 Days and 19.5 Hours
BACKGROUND
The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term in vivo and offspring analysis; therefore, suitable for both medicine and animal production.
AIM
To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine.
METHODS
The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation.
RESULTS
The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts.
CONCLUSION
For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.
Core Tip: The porcine induced pluripotent stem cells (piPSCs) derived from urine derived cells (UDCs) may facilitate their routine and large-scale use by avoiding injury or stress during collection for autologous purposes. However, the precise reprogramming process and characterization is not fully elucidated in other species than murine or human. The generation of piPSCs from UDCs can contribute as a biomedical model for regenerative and translational medicine, as well as for animal production and to elucidate the reprogramming process in porcine, a large animal model.
