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Nunes OBDS, Buranello TW, Farias FDA, Rosero J, Recchia K, Bressan FF. Can cell-cultured meat from stem cells pave the way for sustainable alternative protein? Curr Res Food Sci 2025; 10:100979. [PMID: 40040753 PMCID: PMC11878651 DOI: 10.1016/j.crfs.2025.100979] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 01/09/2025] [Accepted: 01/18/2025] [Indexed: 03/06/2025] Open
Abstract
As the global population grows, the demand for food and animal-derived products rises significantly, posing a notable challenge to the progress of society in general. Alternative protein production may adequately address such a challenge, and cell-based meat production emerges as a promising solution. This review investigates methodologies for in vitro myogenesis and adipogenesis from stem cells (adult, embryonic, or induced pluripotent stem cells - iPSCs) across different animal species, as well as the remaining challenges for scalability, the possibility of genetic modification, along with safety concerns regarding the commercialization of cell-cultured meat. Regarding such complexities, interdisciplinary approaches will be vital for assessing the potential of cell-cultured meat as a sustainable protein source, mimicking the sensory and nutritional attributes of conventional livestock meat whilst meeting the demands of a growing global population while mitigating environmental impacts.
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Affiliation(s)
- Octavio Bignardi da Silva Nunes
- Department of Food Engineering, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
| | - Tiago Willian Buranello
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Fabiana de Andrade Farias
- Department of Food Engineering, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
| | - Jenyffer Rosero
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Kaiana Recchia
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
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Hutchinson AM, Appeltant R, Burdon T, Bao Q, Bargaje R, Bodnar A, Chambers S, Comizzoli P, Cook L, Endo Y, Harman B, Hayashi K, Hildebrandt T, Korody ML, Lakshmipathy U, Loring JF, Munger C, Ng AHM, Novak B, Onuma M, Ord S, Paris M, Pask AJ, Pelegri F, Pera M, Phelan R, Rosental B, Ryder OA, Sukparangsi W, Sullivan G, Tay NL, Traylor-Knowles N, Walker S, Weberling A, Whitworth DJ, Williams SA, Wojtusik J, Wu J, Ying QL, Zwaka TP, Kohler TN. Advancing stem cell technologies for conservation of wildlife biodiversity. Development 2024; 151:dev203116. [PMID: 39382939 PMCID: PMC11491813 DOI: 10.1242/dev.203116] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/10/2024]
Abstract
Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.
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Affiliation(s)
| | - Ruth Appeltant
- Gamete Research Centre, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium
| | - Tom Burdon
- The Roslin Institute, RDSVS, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK
| | - Qiuye Bao
- IMCB-ESCAR, A*STAR, 61 Biopolis Drive, Proteos, 138673Singapore
| | | | - Andrea Bodnar
- Gloucester Marine Genomics Institute, 417 Main St, Gloucester, MA 01930, USA
| | - Stuart Chambers
- Brightfield Therapeutics, South San Francisco, CA 94080, USA
| | - Pierre Comizzoli
- Smithsonian National Zoo and Conservation Biology Institute, 3001 Connecticut Ave., NW Washington, DC 20008, USA
| | - Laura Cook
- Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA 94720, USA
| | - Yoshinori Endo
- University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093, USA
| | - Bob Harman
- Vet-Stem Inc. & Personalized Stem Cells, Inc., 14261 Danielson Street, Poway, CA 92064, USA
| | | | - Thomas Hildebrandt
- Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany
| | - Marisa L. Korody
- San Diego Zoo Wildlife Alliance, 2920 Zoo Dr, San Diego, CA 92101, USA
| | - Uma Lakshmipathy
- Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA 02451, USA
| | - Jeanne F. Loring
- The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037, USA
| | - Clara Munger
- Department of Biochemistry, University of Cambridge, Hopkins Building, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK
| | - Alex H. M. Ng
- GC Therapeutics, 610 Main St., North Cambridge, MA 02139, USA
| | - Ben Novak
- Revive & Restore, 1505 Bridgeway, Suite 203, Sausalito, CA 94965, USA
| | - Manabu Onuma
- National Institute for Environmental Studies, 16-2 Onogawa, City of Tsukuba, Ibaraki 305-8506, Japan
| | - Sara Ord
- Colossal Biosciences, 1401 Lavaca St, Unit #155 Austin, TX 78701, USA
| | - Monique Paris
- IBREAM (Institute for Breeding Rare and Endangered African Mammals), Edinburgh EH3 6AT, UK
| | | | - Francisco Pelegri
- University of Wisconsin-Madison, 500 Lincoln Dr, Madison, WI 53706, USA
| | - Martin Pera
- Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
| | - Ryan Phelan
- Revive & Restore, 1505 Bridgeway, Suite 203, Sausalito, CA 94965, USA
| | - Benyamin Rosental
- The Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Center for Regenerative Medicine and Stem Cells, Ben Gurion University of the Negev, Beer Sheva 8410501, Israel
| | - Oliver A. Ryder
- San Diego Zoo Wildlife Alliance, 2920 Zoo Dr, San Diego, CA 92101, USA
| | - Woranop Sukparangsi
- Department of Biology, Faculty of Science, Burapha University, 169 Long-Had Bangsaen Rd, Saen Suk, Chon Buri District, Chon Buri 20131, Thailand
| | - Gareth Sullivan
- Department of Pediatric Research, Oslo University Hospital, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway
- School of Medicine, University of St Andrews, North Haugh, St Andrews KY16 9TF, UK
| | | | - Nikki Traylor-Knowles
- Rosenstiel School of Marine, Atmospheric, and Earth Science, University of Miami,4600, Rickenbacker Cswy, Key Biscayne, FL 33149, USA
| | - Shawn Walker
- ViaGen Pets & Equine, PO Box 1119, Cedar Park, TX 78613, USA
| | | | - Deanne J. Whitworth
- University of Queensland, Sir Fred Schonell Drive, Brisbane, Queensland, 4072, Australia
| | | | - Jessye Wojtusik
- Omaha's Henry Doorly Zoo & Aquarium, 3701 S 10th St, Omaha, NE 68107, USA
| | - Jun Wu
- University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Qi-Long Ying
- Keck School of Medicine of University of Southern California, 1975 Zonal Ave, Los Angeles, CA 90033, USA
| | - Thomas P. Zwaka
- Department of Cell, Developmental, and Regenerative Biology, and Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Timo N. Kohler
- Department of Biochemistry, University of Cambridge, Hopkins Building, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK
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Zeng R, Huang X, Fu W, Ji W, Cai W, Xu M, Lan D. Construction of Lentiviral Vectors Carrying Six Pluripotency Genes in Yak to Obtain Yak iPSC Cells. Int J Mol Sci 2024; 25:9431. [PMID: 39273379 PMCID: PMC11394755 DOI: 10.3390/ijms25179431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 08/02/2024] [Accepted: 08/19/2024] [Indexed: 09/15/2024] Open
Abstract
Yak is an excellent germplasm resource on the Tibetan Plateau and is able to live in high-altitude areas with hypoxic, cold, and harsh environments. Studies on induced pluripotent stem cells (iPSCs) in large ruminants commonly involve a combination strategy involving six transcription factors, Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 (OSKMNL). This strategy tends to utilize genes from the same species to optimize pluripotency maintenance. In this study, we cloned the six pluripotency genes (OSKMNL) from yak and constructed a multi-cistronic lentiviral vector carrying these genes. This vector efficiently delivered the genes into yak fibroblasts, aiming to promote the reprogramming process. We verified that the treated cells had several pluripotency characteristics, marking the first successful construction of a lentiviral system carrying yak pluripotency genes. This achievement lays the foundation for subsequent establishment of yak iPSCs and holds significant implications for yak-breed improvement and germplasm-resource conservation.
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Affiliation(s)
- Ruilin Zeng
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Xianpeng Huang
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Wei Fu
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Wenhui Ji
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Wenyi Cai
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Meng Xu
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
| | - Daoliang Lan
- College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China
- Key Laboratory of Qinghai-Tibet Plateau Animal Genetic Resource and Utilization, Ministry of Education, Southwest Minzu University, Chengdu 610041, China
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de Castro RCF, Buranello TW, Recchia K, de Souza AF, Pieri NCG, Bressan FF. Emerging Contributions of Pluripotent Stem Cells to Reproductive Technologies in Veterinary Medicine. J Dev Biol 2024; 12:14. [PMID: 38804434 PMCID: PMC11130827 DOI: 10.3390/jdb12020014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2024] [Revised: 04/10/2024] [Accepted: 04/22/2024] [Indexed: 05/29/2024] Open
Abstract
The generation of mature gametes and competent embryos in vitro from pluripotent stem cells has been successfully achieved in a few species, mainly in mice, with recent advances in humans and scarce preliminary reports in other domestic species. These biotechnologies are very attractive as they facilitate the understanding of developmental mechanisms and stages that are generally inaccessible during early embryogenesis, thus enabling advanced reproductive technologies and contributing to the generation of animals of high genetic merit in a short period. Studies on the production of in vitro embryos in pigs and cattle are currently used as study models for humans since they present more similar characteristics when compared to rodents in both the initial embryo development and adult life. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine.
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Affiliation(s)
- Raiane Cristina Fratini de Castro
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil; (R.C.F.d.C.); (T.W.B.); (K.R.)
| | - Tiago William Buranello
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil; (R.C.F.d.C.); (T.W.B.); (K.R.)
| | - Kaiana Recchia
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil; (R.C.F.d.C.); (T.W.B.); (K.R.)
| | - Aline Fernanda de Souza
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil;
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil;
| | - Fabiana Fernandes Bressan
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil; (R.C.F.d.C.); (T.W.B.); (K.R.)
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil;
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Chen L, Tang B, Xie G, Yang R, Zhang B, Wang Y, Zhang Y, Jiang D, Zhang X. Bovine Pluripotent Stem Cells: Current Status and Prospects. Int J Mol Sci 2024; 25:2120. [PMID: 38396797 PMCID: PMC10889747 DOI: 10.3390/ijms25042120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Revised: 02/01/2024] [Accepted: 02/07/2024] [Indexed: 02/25/2024] Open
Abstract
Pluripotent stem cells (PSCs) can differentiate into three germ layers and diverse autologous cell lines. Since cattle are the most commonly used large domesticated animals, an important food source, and bioreactors, great efforts have been made to establish bovine PSCs (bPSCs). bPSCs have great potential in bovine breeding and reproduction, modeling in vitro differentiation, imitating cancer development, and modeling diseases. Currently, bPSCs mainly include bovine embryonic stem cells (bESCs), bovine induced pluripotent stem cells (biPSCs), and bovine expanded potential stem cells (bEPSCs). Establishing stable bPSCs in vitro is a critical scientific challenge, and researchers have made numerous efforts to this end. In this review, the category of PSC pluripotency; the establishment of bESCs, biPSCs, and bEPSCs and its challenges; and the application outlook of bPSCs are discussed, aiming to provide references for future research.
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Affiliation(s)
- Lanxin Chen
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Bo Tang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Guanghong Xie
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Rui Yang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Boyang Zhang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Yueqi Wang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Yan Zhang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Daozhen Jiang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Xueming Zhang
- State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China
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Weeratunga P, Harman RM, Van de Walle GR. Induced pluripotent stem cells from domesticated ruminants and their potential for enhancing livestock production. Front Vet Sci 2023; 10:1129287. [PMID: 36891466 PMCID: PMC9986305 DOI: 10.3389/fvets.2023.1129287] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Accepted: 01/31/2023] [Indexed: 02/22/2023] Open
Abstract
Ruminant livestock, including cattle, sheep, goat, and buffalo, are essential for global food security and serve valuable roles in sustainable agricultural systems. With the limited availability of embryonic stem cells (ESCs) from these species, ruminant induced pluripotent stem cells (iPSCs) and iPSC-like cells provide a valuable research tool for agricultural, veterinary, biomedical, and pharmaceutical applications, as well as for the prospect of translation to human medicine. iPSCs are generated by reprogramming of adult or fetal cells to an ESC-like state by ectopic expression of defined transcription factors. Despite the slow pace the field has evolved in livestock species compared to mice and humans, significant progress has been made over the past 15 years in using different cell sources and reprogramming protocols to generate iPSCs/iPSC-like cells from ruminants. This mini review summarizes the current literature related to the derivation of iPSCs/iPSC-like cells from domesticated ruminants with a focus on reprogramming protocols, characterization, associated limitations, and potential applications in ruminant basic science research and production.
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Affiliation(s)
- Prasanna Weeratunga
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States
| | - Rebecca M Harman
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States
| | - Gerlinde R Van de Walle
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States
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Botigelli RC, Pieri NCG, Bessi BW, Machado LS, Bridi A, de Souza AF, Recchia K, Neto PF, Ross PJ, Bressan FF, Nogueira MFG. Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells. Front Cell Dev Biol 2022; 10:938709. [PMID: 36187479 PMCID: PMC9515551 DOI: 10.3389/fcell.2022.938709] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Accepted: 08/11/2022] [Indexed: 12/03/2022] Open
Abstract
Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed “OSKM” factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.
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Affiliation(s)
- Ramon Cesar Botigelli
- Multiuser Facility (FitoFarmaTec), Department of Pharmacology, Biosciences Institute (IBB), São Paulo State University (UNESP), Botucatu, Brazil
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
- Correspondence: Ramon Cesar Botigelli, ; Marcelo Fábio Gouveia Nogueira,
| | - Naira Carolina Godoy Pieri
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Brendon William Bessi
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Lucas Simões Machado
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
- Paulista School of Medicine (EPM), Laboratory of Neurobiology, Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo, Brazil
| | - Alessandra Bridi
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Aline Fernanda de Souza
- Laboratory Biomedical Science, Department of Biomedical Science, Ontario Veterinary College (OVC), University of Guelph, Guelph, ON, Canada
| | - Kaiana Recchia
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Paulo Fantinato Neto
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Pablo Juan Ross
- Laboratory Department of Animal Science, University of California, Davis, Davis, CA, United States
| | - Fabiana Fernandes Bressan
- Laboratory of Molecular Morphophysiology and Development (LMMD), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, Brazil
| | - Marcelo Fábio Gouveia Nogueira
- Multiuser Facility (FitoFarmaTec), Department of Pharmacology, Biosciences Institute (IBB), São Paulo State University (UNESP), Botucatu, Brazil
- School of Sciences and Languages, Laboratory of Embryonic Micromanipulation, Department of Biological Sciences, São Paulo State University (UNESP), Assis, Brazil
- Correspondence: Ramon Cesar Botigelli, ; Marcelo Fábio Gouveia Nogueira,
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Recchia K, Pessôa LVDF, Pieri NCG, Pires PRL, Bressan FF. Influence of Cell Type in In Vitro Induced Reprogramming in Cattle. Life (Basel) 2022; 12:1139. [PMID: 36013318 PMCID: PMC9409886 DOI: 10.3390/life12081139] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 07/16/2022] [Accepted: 07/24/2022] [Indexed: 06/15/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro.
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Affiliation(s)
- Kaiana Recchia
- Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo 05508-270, SP, Brazil
| | - Laís Vicari de Figueiredo Pessôa
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil; (L.V.d.F.P.); (N.C.G.P.); (P.R.L.P.)
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil; (L.V.d.F.P.); (N.C.G.P.); (P.R.L.P.)
| | - Pedro Ratto Lisboa Pires
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil; (L.V.d.F.P.); (N.C.G.P.); (P.R.L.P.)
| | - Fabiana Fernandes Bressan
- Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo 05508-270, SP, Brazil
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil; (L.V.d.F.P.); (N.C.G.P.); (P.R.L.P.)
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Fang L, Feng Z, Mei J, Zhou J, Lin Z. [Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro]. NAN FANG YI KE DA XUE XUE BAO = JOURNAL OF SOUTHERN MEDICAL UNIVERSITY 2022; 42:929-936. [PMID: 35790445 DOI: 10.12122/j.issn.1673-4254.2022.06.18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
OBJECTIVE To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
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Affiliation(s)
- L Fang
- Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China
| | - Z Feng
- Ji Hua Institute of Biomedical Engineering Technology, Ji Hua Laboratory, Foshan 528200, China
| | - J Mei
- School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
| | - J Zhou
- Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China
| | - Z Lin
- Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China
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Recchia K, Machado LS, Botigelli RC, Pieri NCG, Barbosa G, de Castro RVG, Marques MG, Pessôa LVDF, Fantinato Neto P, Meirelles FV, Souza AFD, Martins SMMK, Bressan FF. In vitro induced pluripotency from urine-derived cells in porcine. World J Stem Cells 2022; 14:231-244. [PMID: 35432738 PMCID: PMC8968213 DOI: 10.4252/wjsc.v14.i3.231] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 10/11/2021] [Accepted: 02/16/2022] [Indexed: 02/06/2023] Open
Abstract
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.
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Affiliation(s)
- Kaiana Recchia
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Lucas Simões Machado
- 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
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Gabriela Barbosa
- 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
| | - Laís Vicari de Figueiredo Pessôa
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Paulo Fantinato Neto
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Flávio Vieira Meirelles
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Aline Fernanda de Souza
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | | | - Fabiana Fernandes Bressan
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
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