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Olenic M, Deelkens C, Heyman E, De Vlieghere E, Zheng X, van Hengel J, De Schauwer C, Devriendt B, De Smet S, Thorrez L. Review: Livestock cell types with myogenic differentiation potential: Considerations for the development of cultured meat. Animal 2025; 19 Suppl 1:101242. [PMID: 39097434 DOI: 10.1016/j.animal.2024.101242] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2023] [Revised: 06/27/2024] [Accepted: 07/02/2024] [Indexed: 08/05/2024] Open
Abstract
With the current environmental impact of large-scale animal production and societal concerns about the welfare of farm animals, researchers are questioning whether we can cultivate animal cells for the purpose of food production. This review focuses on a pivotal aspect of the cellular agriculture domain: cells. We summarised information on the various cell types from farm animals currently used for the development of cultured meat, including mesenchymal stromal cells, myoblasts, and pluripotent stem cells. The review delves into the advantages and limitations of each cell type and considers factors like the selection of the appropriate cell source, as well as cell culture conditions that influence cell performance. As current research in cultured meat seeks to create muscle fibers to mimic the texture and nutritional profile of meat, we focused on the myogenic differentiation capacity of the cells. The most commonly used cell type for this purpose are myoblasts or satellite cells, but given their limited proliferation capacity, efforts are underway to formulate myogenic differentiation protocols for mesenchymal stromal cells and pluripotent stem cells. The multipotent character of the latter cell types might enable the creation of other tissues found in meat, such as adipose and connective tissues. This review can help guiding the selection of a cell type or culture conditions in the context of cultured meat development.
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Affiliation(s)
- M Olenic
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven Campus Kulak, Kortrijk, Belgium; Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - C Deelkens
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven Campus Kulak, Kortrijk, Belgium; Medical Cell Biology, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
| | - E Heyman
- Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - E De Vlieghere
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven Campus Kulak, Kortrijk, Belgium; Polymer Chemistry and Biomaterials Group, Centre of Macromolecular Chemistry, Ghent University, Belgium
| | - X Zheng
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven Campus Kulak, Kortrijk, Belgium
| | - J van Hengel
- Medical Cell Biology, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
| | - C De Schauwer
- Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - B Devriendt
- Laboratory of Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - S De Smet
- Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
| | - L Thorrez
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven Campus Kulak, Kortrijk, Belgium.
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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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Wu Y, Wang C, Fan X, Ma Y, Liu Z, Ye X, Shen C, Wu C. The impact of induced pluripotent stem cells in animal conservation. Vet Res Commun 2024; 48:649-663. [PMID: 38228922 DOI: 10.1007/s11259-024-10294-3] [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: 11/02/2023] [Accepted: 01/04/2024] [Indexed: 01/18/2024]
Abstract
It is widely acknowledged that we are currently facing a critical tipping point with regards to global extinction, with human activities driving us perilously close to the brink of a devastating sixth mass extinction. As a promising option for safeguarding endangered species, induced pluripotent stem cells (iPSCs) hold great potential to aid in the preservation of threatened animal populations. For endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni), supply of embryos is often limited. After the death of the last male in 2019, only two females remained in the world. IPSC technology offers novel approaches and techniques for obtaining pluripotent stem cells (PSCs) from rare and endangered animal species. Successful generation of iPSCs circumvents several bottlenecks that impede the development of PSCs, including the challenges associated with establishing embryonic stem cells, limited embryo sources and immune rejection following embryo transfer. To provide more opportunities and room for growth in our work on animal welfare, in this paper we will focus on the progress made with iPSC lines derived from endangered and extinct species, exploring their potential applications and limitations in animal welfare research.
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Affiliation(s)
- Yurou Wu
- School of Pharmacy/School of Modem Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People's Republic of China
| | - Chengwei Wang
- School of Pharmacy/School of Modem Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People's Republic of China
| | - Xinyun Fan
- School of Pharmacy/School of Modem Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People's Republic of China
| | - Yuxiao Ma
- Department of Biology, New York University, New York, NY, USA
| | - Zibo Liu
- School of Pharmacy/School of Modem Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People's Republic of China
| | - Xun Ye
- School of Pharmacy/School of Modem Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People's Republic of China
| | - Chongyang Shen
- School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, 611137, People's Republic of China.
| | - Chunjie Wu
- Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu Univesity of Traditional Chinese Medicine, Chengdu, Sichuan, 611137, People's Republic of China.
- Sichuan Engineering Research Center for Endangered Medicinal Animals, Chengdu, 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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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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Liu M, Zhao L, Wang Z, Su H, Wang T, Yang G, Chen L, Wu B, Zhao G, Guo J, Yang Z, Zhang J, Hao C, Ma T, Song Y, Bao S, Zuo Y, Li X, Cao G. Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition. Front Cell Dev Biol 2021; 9:785055. [PMID: 34977028 PMCID: PMC8716767 DOI: 10.3389/fcell.2021.785055] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Accepted: 11/12/2021] [Indexed: 11/18/2022] Open
Abstract
Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.
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Affiliation(s)
- Moning Liu
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
| | - Lixia Zhao
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
- Inner Mongolia Saikexing Institutes of Breeding and Reproductive Biotechnologies in Domestic Animal, Hohhot, China
| | - Zixin Wang
- Inner Mongolia Saikexing Institutes of Breeding and Reproductive Biotechnologies in Domestic Animal, Hohhot, China
| | - Hong Su
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
| | - Tong Wang
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
| | - Guang Yang
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Lu Chen
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
| | - Baojiang Wu
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Gaoping Zhao
- Inner Mongolia Saikexing Institutes of Breeding and Reproductive Biotechnologies in Domestic Animal, Hohhot, China
| | - Jitong Guo
- Inner Mongolia Saikexing Institutes of Breeding and Reproductive Biotechnologies in Domestic Animal, Hohhot, China
| | - Zhiqing Yang
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Jia Zhang
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Chunxia Hao
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Teng Ma
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
| | - Yongli Song
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Siqin Bao
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yongchun Zuo
- China State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Xihe Li
- Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
- Inner Mongolia Saikexing Institutes of Breeding and Reproductive Biotechnologies in Domestic Animal, Hohhot, China
- *Correspondence: Guifang Cao, ; Xihe Li,
| | - Guifang Cao
- Inner Mongolia Key Laboratory of Basic Veterinary Medicine, College of Veterinary, Inner Mongolia Agricultural University, Hohhot, China
- *Correspondence: Guifang Cao, ; Xihe Li,
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Martínez-Falguera D, Iborra-Egea O, Gálvez-Montón C. iPSC Therapy for Myocardial Infarction in Large Animal Models: Land of Hope and Dreams. Biomedicines 2021; 9:1836. [PMID: 34944652 PMCID: PMC8698445 DOI: 10.3390/biomedicines9121836] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Revised: 11/30/2021] [Accepted: 12/01/2021] [Indexed: 02/07/2023] Open
Abstract
Myocardial infarction is the main driver of heart failure due to ischemia and subsequent cell death, and cell-based strategies have emerged as promising therapeutic methods to replace dead tissue in cardiovascular diseases. Research in this field has been dramatically advanced by the development of laboratory-induced pluripotent stem cells (iPSCs) that harbor the capability to become any cell type. Like other experimental strategies, stem cell therapy must meet multiple requirements before reaching the clinical trial phase, and in vivo models are indispensable for ensuring the safety of such novel therapies. Specifically, translational studies in large animal models are necessary to fully evaluate the therapeutic potential of this approach; to empirically determine the optimal combination of cell types, supplementary factors, and delivery methods to maximize efficacy; and to stringently assess safety. In the present review, we summarize the main strategies employed to generate iPSCs and differentiate them into cardiomyocytes in large animal species; the most critical differences between using small versus large animal models for cardiovascular studies; and the strategies that have been pursued regarding implanted cells' stage of differentiation, origin, and technical application.
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Affiliation(s)
- Daina Martínez-Falguera
- Faculty of Medicine, University of Barcelona (UB), 08036 Barcelona, Spain;
- ICREC Research Program, Germans Trias i Pujol Health Research Institute, Can Ruti Campus, 08916 Badalona, Spain;
- Heart Institute (iCor), Germans Trias i Pujol University Hospital, 08916 Badalona, Spain
| | - Oriol Iborra-Egea
- ICREC Research Program, Germans Trias i Pujol Health Research Institute, Can Ruti Campus, 08916 Badalona, Spain;
- Heart Institute (iCor), Germans Trias i Pujol University Hospital, 08916 Badalona, Spain
| | - Carolina Gálvez-Montón
- ICREC Research Program, Germans Trias i Pujol Health Research Institute, Can Ruti Campus, 08916 Badalona, Spain;
- Heart Institute (iCor), Germans Trias i Pujol University Hospital, 08916 Badalona, Spain
- CIBERCV, Instituto de Salud Carlos III, 28029 Madrid, Spain
- Institut d’Investigació Biomèdica de Bellvitge-IDIBELL, L’Hospitalet de Llobregat, 08908 Barcelona, Spain
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8
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Thin films of functionalized carbon nanotubes support long-term maintenance and cardio-neuronal differentiation of canine induced pluripotent stem cells. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2021; 40:102487. [PMID: 34740869 DOI: 10.1016/j.nano.2021.102487] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Accepted: 03/25/2021] [Indexed: 01/24/2023]
Abstract
Induced pluripotent stem cells (iPSCs) are a promising cell source for regenerative medicine. However, their feeder-free maintenance in undifferentiated states remains challenging. In recent past extensive studies have been directed using pristine or functionalized carbon nanotube in tissue engineering. Here we proposed thin films of functionalized carbon nanotubes (OH-single-walled CNTs [SWCNTs] and OH-multiwalled CNTs [MWCNTs]), as alternatives for the feeder-free in vitro culture of canine iPSCs (ciPSCs), considered as the cellular model. The ciPSC colonies could maintain their dome-shaped compactness and other characteristics when propagated on CNT films. Concomitantly, high cell viability and upregulation of pluripotency-associated genes and cell adhesion molecules were observed, further supported by molecular docking. Moreover, CNTs did not have profound toxic effects compared to feeder cultures as evident by cytocompatibility studies. Further, cardiac and neuronal differentiation of ciPSCs was induced on these films to determine their influence on the differentiation process. The cells retained differentiation potential and the nanotopographical features of the substrates provided positive cues to enhance differentiation to both lineages as evident by immunocytochemical staining and marker gene expression. Overall, OH-SWCNT provided better cues, maintained pluripotency, and induced the differentiation of ciPSCs. These results indicate that OH-functionalized CNT films could be used as alternatives for the feeder-free maintenance of ciPSCs towards prospective utilization in regenerative medicine.
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Pillai VV, Koganti PP, Kei TG, Gurung S, Butler WR, Selvaraj V. Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells. Biol Open 2021; 10:272681. [PMID: 34719702 PMCID: PMC8565620 DOI: 10.1242/bio.058756] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Accepted: 09/14/2021] [Indexed: 12/13/2022] Open
Abstract
Although derivation of naïve bovine embryonic stem cells is unachieved, the possibility for generation of bovine induced pluripotent stem cells (biPSCs) has been generally reported. However, attempts to sustain biPSCs by promoting self-renewal have not been successful. Methods established for maintaining murine and human induced pluripotent stem cells (iPSCs) do not support self-renewal of iPSCs for any bovid species. In this study, we examined methods to enhance complete reprogramming and concurrently investigated signaling relevant to pluripotency of the bovine blastocyst inner cell mass (ICM). First, we identified that forced expression of SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) substantially enhanced the reprogramming efficacy of bovine fibroblasts to biPSCs. Second, we uncovered that TGFβ signaling is actively perturbed in the ICM. Inhibition of ALK4/5/7 to block TGFβ/activin/nodal signaling together with GSK3β and MEK1/2 supported robust in vitro self-renewal of naïve biPSCs with unvarying colony morphology, steady expansion, expected pluripotency gene expression and committed differentiation plasticity. Core similarities between biPSCs and stem cells of the 16-cell-stage bovine embryo indicated a stable ground state of pluripotency; this allowed us to reliably gain predictive understanding of signaling in bovine pluripotency using systems biology approaches. Beyond defining a high-fidelity platform for advancing biPSC-based biotechnologies that have not been previously practicable, these findings also represent a significant step towards understanding corollaries and divergent aspects of bovine pluripotency. This article has an associated First Person interview with the joint first authors of the paper. Summary: Pluripotency reprogramming by overcoming the stable epigenome of bovine cells, and uncovering precise early embryo self-renewal mechanisms enables sustenance and expansion of authentic induced pluripotent stem cells in vitro.
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Affiliation(s)
- Viju Vijayan Pillai
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
| | - Prasanthi P Koganti
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
| | - Tiffany G Kei
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
| | - Shailesh Gurung
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
| | - W Ronald Butler
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
| | - Vimal Selvaraj
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853, USA
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Su Y, Wang L, Fan Z, Liu Y, Zhu J, Kaback D, Oudiz J, Patrick T, Yee SP, Tian X(C, Polejaeva I, Tang Y. Establishment of Bovine-Induced Pluripotent Stem Cells. Int J Mol Sci 2021; 22:ijms221910489. [PMID: 34638830 PMCID: PMC8508593 DOI: 10.3390/ijms221910489] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 09/04/2021] [Accepted: 09/24/2021] [Indexed: 11/22/2022] Open
Abstract
Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.
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Affiliation(s)
- Yue Su
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
| | - Ling Wang
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
| | - Zhiqiang Fan
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA; (Z.F.); (Y.L.); (T.P.)
| | - Ying Liu
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA; (Z.F.); (Y.L.); (T.P.)
| | - Jiaqi Zhu
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
| | - Deborah Kaback
- Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA; (D.K.); (S.P.Y.)
| | - Julia Oudiz
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
| | - Tayler Patrick
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA; (Z.F.); (Y.L.); (T.P.)
| | - Siu Pok Yee
- Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA; (D.K.); (S.P.Y.)
| | - Xiuchun (Cindy) Tian
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
| | - Irina Polejaeva
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA; (Z.F.); (Y.L.); (T.P.)
- Correspondence: (I.P.); (Y.T.)
| | - Young Tang
- Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT 06268, USA; (Y.S.); (L.W.); (J.Z.); (J.O.); (X.T.)
- Correspondence: (I.P.); (Y.T.)
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11
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Bessi BW, Botigelli RC, Pieri NCG, Machado LS, Cruz JB, de Moraes P, de Souza AF, Recchia K, Barbosa G, de Castro RVG, Nogueira MFG, Bressan FF. Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation. Cells 2021; 10:cells10061531. [PMID: 34204517 PMCID: PMC8234940 DOI: 10.3390/cells10061531] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 06/07/2021] [Accepted: 06/15/2021] [Indexed: 12/11/2022] Open
Abstract
The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i-bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.
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Affiliation(s)
- Brendon Willian Bessi
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Ramon Cesar Botigelli
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
- Department of Pharmacology, Institute of Biosciences (IBB), São Paulo State University (UNESP), Botucatu 18618-689, Brazil
- Correspondence: (R.C.B.); (F.F.B.)
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo (USP), São Paulo 05508-270, Brazil
| | - Lucas Simões Machado
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Jessica Brunhara Cruz
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Pamela de Moraes
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Aline Fernanda de Souza
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Kaiana Recchia
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Gabriela Barbosa
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
| | - Raquel Vasconcelos Guimarães de Castro
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
- Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Botucatu 14884-900, Brazil
| | - Marcelo Fábio Gouveia Nogueira
- Department of Biological Science, School of Sciences, Humanities and Languages, São Paulo State University (UNESP), Assis 19806-900, Brazil;
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (B.W.B.); (N.C.G.P.); (L.S.M.); (J.B.C.); (P.d.M.); (A.F.d.S.); (K.R.); (G.B.); (R.V.G.d.C.)
- Correspondence: (R.C.B.); (F.F.B.)
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12
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Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle. Int J Mol Sci 2021; 22:ijms22095011. [PMID: 34065074 PMCID: PMC8125899 DOI: 10.3390/ijms22095011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 05/07/2021] [Accepted: 05/07/2021] [Indexed: 12/29/2022] Open
Abstract
Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.
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13
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Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.
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14
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Xiang J, Wang H, Zhang Y, Wang J, Liu F, Han X, Lu Z, Li C, Li Z, Gao Y, Tian Y, Wang Y, Li X. LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine-mouse chimeras from iPSCs and bovine fetal fibroblasts. FEBS J 2021; 288:4394-4411. [PMID: 33524211 DOI: 10.1111/febs.15744] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 12/19/2020] [Accepted: 01/29/2021] [Indexed: 12/15/2022]
Abstract
Cattle have emerged as one of the most important domestic animals widely used for meat, milk, and fur. Derivation of bovine pluripotent stem cells (PSCs) can be applied in drug selecting and human disease modeling and facilitated agriculture-related applications such as production of genetically excellent cattle by gene editing. Extended PSCs (EPSCs), capable of differentiating into embryonic and extraembryonic parts, have been generated in mouse, human, and pig. Whether bovine EPSCs could be generated, and their chimeric competency remains unclear. This study focused on derivation of bovine EPSCs using LCDM medium and exploring the characteristics of EPSCs among different species, including bovine, mouse, and human EPSCs. Here, using LCDM medium (consisting of hLIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) enables the derivation of bovine EPSCs from induced PSCs (iPSCs) and bovine fetal fibroblasts (BFF) with stable morphology, pluripotent marker expression, and in vitro differentiation ability. Notably, bovine EPSCs exhibited interspecies chimeric contribution to embryonic and extraembryonic tissues in pre-implantation blastocysts and postimplantation bovine-mouse chimeras. Transcriptome analysis revealed the unique molecular characteristics of bovine EPSCs compared with iPSCs. The similarities and differences in molecular features across bovine, human, and mouse EPSCs were also described by transcriptome analysis. Taken together, the LCDM culture system containing chemical cocktails can be used for the establishment and long-term passaging of bovine EPSCs with embryonic and extraembryonic potency in bovine-mouse chimeras. Our findings lay the foundation of generating PSCs in domestic animals and open avenues for basic and applied research in biology, medicine, and agriculture. DATABASE: Gene expression data of bovine EPSCs and bovine iPSCs are available in the GEO databases under the accession number PRJNA693452.
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Affiliation(s)
- Jinzhu Xiang
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Hanning Wang
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yuanyuan Zhang
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Jing Wang
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Fang Liu
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Xuejie Han
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Zhenyu Lu
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Chen Li
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Zihong Li
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yanru Gao
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yujing Tian
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yingjie Wang
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Xueling Li
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China
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15
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Su Y, Zhu J, Salman S, Tang Y. Induced pluripotent stem cells from farm animals. J Anim Sci 2021; 98:5937369. [PMID: 33098420 DOI: 10.1093/jas/skaa343] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023] Open
Abstract
The development of the induced pluripotent stem cells (iPSCs) technology has revolutionized the world on the establishment of pluripotent stem cells (PSCs) across a great variety of animal species. Generation of iPSCs from domesticated animals would provide unrestricted cell resources for the study of embryonic development and cell differentiation of these species, for screening and establishing desired traits for sustainable agricultural production, and as veterinary and preclinical therapeutic tools for animal and human diseases. Induced PSCs from domesticated animals thus harbor enormous scientific, economical, and societal values. Although much progress has been made toward the generation of PSCs from these species, major obstacles remain precluding the exclamation of the establishment of bona fide iPSCs. The most prominent of them remain the inability of these cells to silence exogenous reprogramming factors, the obvious reliance on exogenous factors for their self-renewal, and the restricted development potential in vivo. In this review, we summarize the history and current progress in domestic farm animal iPSC generation, with a focus on swine, ruminants (cattle, ovine, and caprine), horses, and avian species (quails and chickens). We also discuss the problems associated with the farm animal iPSCs and potential future directions toward the complete reprogramming of somatic cells from farm animals.
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Affiliation(s)
- Yue Su
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Jiaqi Zhu
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Saleh Salman
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Young Tang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
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16
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Kumar D, Talluri TR, Selokar NL, Hyder I, Kues WA. Perspectives of pluripotent stem cells in livestock. World J Stem Cells 2021; 13:1-29. [PMID: 33584977 PMCID: PMC7859985 DOI: 10.4252/wjsc.v13.i1.1] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/28/2020] [Accepted: 11/09/2020] [Indexed: 02/06/2023] Open
Abstract
The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.
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Affiliation(s)
- Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India.
| | - Thirumala R Talluri
- Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner 334001, India
| | - Naresh L Selokar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India
| | - Iqbal Hyder
- Department of Physiology, NTR College of Veterinary Science, Gannavaram 521102, India
| | - Wilfried A Kues
- Department of Biotechnology, Friedrich-Loeffler-Institute, Federal Institute of Animal Health, Neustadt 31535, Germany
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17
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Scarfone RA, Pena SM, Russell KA, Betts DH, Koch TG. The use of induced pluripotent stem cells in domestic animals: a narrative review. BMC Vet Res 2020; 16:477. [PMID: 33292200 PMCID: PMC7722595 DOI: 10.1186/s12917-020-02696-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 11/24/2020] [Indexed: 02/07/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) are undifferentiated stem cells characterized by the ability to differentiate into any cell type in the body. iPSCs are a relatively new and rapidly developing technology in many fields of biology, including developmental anatomy and physiology, pathology, and toxicology. These cells have great potential in research as they are self-renewing and pluripotent with minimal ethical concerns. Protocols for their production have been developed for many domestic animal species, which have since been used to further our knowledge in the progression and treatment of diseases. This research is valuable both for veterinary medicine as well as for the prospect of translation to human medicine. Safety, cost, and feasibility are potential barriers for this technology that must be considered before widespread clinical adoption. This review will analyze the literature pertaining to iPSCs derived from various domestic species with a focus on iPSC production and characterization, applications for tissue and disease research, and applications for disease treatment.
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Affiliation(s)
- Rachel A Scarfone
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Samantha M Pena
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Keith A Russell
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Dean H Betts
- Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada
| | - Thomas G Koch
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada.
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18
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Navarro M, Soto DA, Pinzon CA, Wu J, Ross PJ. Livestock pluripotency is finally captured in vitro. Reprod Fertil Dev 2020; 32:11-39. [PMID: 32188555 DOI: 10.1071/rd19272] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Pluripotent stem cells (PSCs) have demonstrated great utility in improving our understanding of mammalian development and continue to revolutionise regenerative medicine. Thanks to the improved understanding of pluripotency in mice and humans, it has recently become feasible to generate stable livestock PSCs. Although it is unlikely that livestock PSCs will be used for similar applications as their murine and human counterparts, new exciting applications that could greatly advance animal agriculture are being developed, including the use of PSCs for complex genome editing, cellular agriculture, gamete generation and invitro breeding schemes.
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Affiliation(s)
- Micaela Navarro
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA 95616, USA
| | - Delia A Soto
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA 95616, USA
| | - Carlos A Pinzon
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA; and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Pablo J Ross
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA 95616, USA; and Corresponding author.
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19
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Mavaro I, De Felice E, Palladino A, D'Angelo L, de Girolamo P, Attanasio C. Anatomical templates for tissue (re)generation and beyond. Biotechnol Bioeng 2020; 117:3938-3951. [PMID: 32776516 DOI: 10.1002/bit.27533] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Revised: 07/27/2020] [Accepted: 08/07/2020] [Indexed: 11/12/2022]
Abstract
Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4. Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as "cellular agriculture." iPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely "cultured meat," a product that improves animal welfare and encourages the consumption of healthier meat along with environmental preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species.
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Affiliation(s)
- Isabella Mavaro
- Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy.,Interdepartmental Center for Research in Biomaterials (CRIB), University of Naples Federico II, Naples, Italy
| | - Elena De Felice
- School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy
| | - Antonio Palladino
- Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy
| | - Livia D'Angelo
- Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy.,Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy
| | - Paolo de Girolamo
- Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy
| | - Chiara Attanasio
- Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy.,Interdepartmental Center for Research in Biomaterials (CRIB), University of Naples Federico II, Naples, Italy.,Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Naples, Italy
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20
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Bressan FF, Bassanezze V, de Figueiredo Pessôa LV, Sacramento CB, Malta TM, Kashima S, Fantinato Neto P, Strefezzi RDF, Pieri NCG, Krieger JE, Covas DT, Meirelles FV. Generation of induced pluripotent stem cells from large domestic animals. Stem Cell Res Ther 2020; 11:247. [PMID: 32586372 PMCID: PMC7318412 DOI: 10.1186/s13287-020-01716-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 03/23/2020] [Accepted: 05/07/2020] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Induced pluripotent stem cells (iPSCs) have enormous potential in developmental biology studies and in cellular therapies. Although extensively studied and characterized in human and murine models, iPSCs from animals other than mice lack reproducible results. METHODS Herein, we describe the generation of robust iPSCs from equine and bovine cells through lentiviral transduction of murine or human transcription factors Oct4, Sox2, Klf4, and c-Myc and from human and murine cells using similar protocols, even when different supplementations were used. The iPSCs were analyzed regarding morphology, gene and protein expression of pluripotency factors, alkaline phosphatase detection, and spontaneous and induced differentiation. RESULTS Although embryonic-derived stem cells are yet not well characterized in domestic animals, generation of iPS cells from these species is possible through similar protocols used for mouse or human cells, enabling the use of pluripotent cells from large animals for basic or applied purposes. Herein, we also infer that bovine iPS (biPSCs) exhibit similarity to mouse iPSCs (miPSCs), whereas equine iPSs (eiPSCs) to human (hiPSCs). CONCLUSIONS The generation of reproducible protocols in different animal species will provide an informative tool for producing in vitro autologous pluripotent cells from domestic animals. These cells will create new opportunities in animal breeding through transgenic technology and will support a new era of translational medicine with large animal models.
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Affiliation(s)
- Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
- Postgraduate Program in Anatomy of Domestic and Wild Animals, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
- Center for Cell-Based Therapy, Regional Blood Center, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Vinícius Bassanezze
- Heart Institute (INCOR), Faculty of Medicine, University of São Paulo, São Paulo, Brazil
- Present Address: Brigham and Women’s Hospital, Harvard Medical School, Boston, USA
| | - Laís Vicari de Figueiredo Pessôa
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
- Postgraduate Program in Anatomy of Domestic and Wild Animals, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Chester Bittencourt Sacramento
- Heart Institute (INCOR), Faculty of Medicine, University of São Paulo, São Paulo, Brazil
- Present Address: Weill Cornell Medicine, Cornell University, Ithaca, USA
| | - Tathiane Maistro Malta
- Center for Cell-Based Therapy, Regional Blood Center, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
- School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP Brazil
| | - Simone Kashima
- Center for Cell-Based Therapy, Regional Blood Center, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Paulo Fantinato Neto
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
- Postgraduate Program in Anatomy of Domestic and Wild Animals, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Ricardo De Francisco Strefezzi
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - José Eduardo Krieger
- Heart Institute (INCOR), Faculty of Medicine, University of São Paulo, São Paulo, Brazil
| | - Dimas Tadeu Covas
- Center for Cell-Based Therapy, Regional Blood Center, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Flávio Vieira Meirelles
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
- Postgraduate Program in Anatomy of Domestic and Wild Animals, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
- Center for Cell-Based Therapy, Regional Blood Center, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
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21
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Li J, Wang T, Li C, Wang Z, Wang P, Zheng L. Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model. J Cell Physiol 2020; 235:6754-6766. [PMID: 32012286 DOI: 10.1002/jcp.29569] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Accepted: 01/09/2020] [Indexed: 12/12/2022]
Abstract
Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.
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Affiliation(s)
- Jingyi Li
- Department of Medical Cosmetology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China
| | - Tingting Wang
- Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Chong Li
- Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Zhifang Wang
- Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Peng Wang
- Department of Comprehensive Surgery, Peking University Third Hospital, Beijing, China
| | - Lili Zheng
- Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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22
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Wang J, Xue X, Fan K, Liu Q, Zhang S, Peng M, Zhou J, Cao Z. Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels. Theriogenology 2019; 145:149-157. [PMID: 31733931 DOI: 10.1016/j.theriogenology.2019.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 09/25/2019] [Accepted: 10/09/2019] [Indexed: 12/25/2022]
Abstract
The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.
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Affiliation(s)
- Juhua Wang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding in Anhui Provincial, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China.
| | - Xiuheng Xue
- College of Tea & Food Science and Technology, Anhui Agricultural University, Hefei, China.
| | - Kai Fan
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Qi Liu
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Suzi Zhang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Mengling Peng
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China
| | - Jie Zhou
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China
| | - Zubing Cao
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding in Anhui Provincial, Hefei, China
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23
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Rawat N, Singh MK, Sharma T, Vats P, Nagoorvali D, Palta P, Chauhan MS, Manik RS. Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts. Anim Biotechnol 2019; 32:155-168. [PMID: 31599201 DOI: 10.1080/10495398.2019.1671435] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.
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Affiliation(s)
- Nidhi Rawat
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manoj Kumar Singh
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Tushar Sharma
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Preeti Vats
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - D Nagoorvali
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Prabhat Palta
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manmohan Singh Chauhan
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India.,ICAR-Central Institute for Research on Goats, Makhdum, India
| | - Radhey Sham Manik
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
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24
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Pessôa LVDF, Bressan FF, Freude KK. Induced pluripotent stem cells throughout the animal kingdom: Availability and applications. World J Stem Cells 2019; 11:491-505. [PMID: 31523369 PMCID: PMC6716087 DOI: 10.4252/wjsc.v11.i8.491] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2019] [Revised: 06/18/2019] [Accepted: 06/20/2019] [Indexed: 02/06/2023] Open
Abstract
Up until the mid 2000s, the capacity to generate every cell of an organism was exclusive to embryonic stem cells. In 2006, researchers Takahashi and Yamanaka developed an alternative method of generating embryonic-like stem cells from adult cells, which they coined induced pluripotent stem cells (iPSCs). Such iPSCs possess most of the advantages of embryonic stem cells without the ethical stigma associated with derivation of the latter. The possibility of generating “custom-made” pluripotent cells, ideal for patient-specific disease models, alongside their possible applications in regenerative medicine and reproduction, has drawn a lot of attention to the field with numbers of iPSC studies published growing exponentially. IPSCs have now been generated for a wide variety of species, including but not limited to, mouse, human, primate, wild felines, bovines, equines, birds and rodents, some of which still lack well-established embryonic stem cell lines. The paucity of robust characterization of some of these iPSC lines as well as the residual expression of transgenes involved in the reprogramming process still hampers the use of such cells in species preservation or medical research, underscoring the requirement for further investigations. Here, we provide an extensive overview of iPSC generated from a broad range of animal species including their potential applications and limitations.
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Affiliation(s)
- Laís Vicari de Figueiredo Pessôa
- Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg 1870, Denmark
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Kristine Karla Freude
- Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg 1870, Denmark
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25
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Pillai VV, Kei TG, Reddy SE, Das M, Abratte C, Cheong SH, Selvaraj V. Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance. Anim Sci J 2019; 90:1149-1160. [PMID: 31322312 DOI: 10.1111/asj.13272] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Revised: 05/10/2019] [Accepted: 06/17/2019] [Indexed: 12/16/2022]
Abstract
Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC-like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC-driven bovine technologies can be realized.
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Affiliation(s)
- Viju V Pillai
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
| | - Tiffany G Kei
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
| | - Shannon E Reddy
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
| | - Moubani Das
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
| | - Christian Abratte
- iPSC Core Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Soon H Cheong
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Vimal Selvaraj
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
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26
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Kumar D, Anand T, Vijayalakshmy K, Sharma P, Rajendran R, Selokar NL, Yadav PS, Kumar D. Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions. Res Vet Sci 2019; 123:252-260. [PMID: 30703616 DOI: 10.1016/j.rvsc.2019.01.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Revised: 01/14/2019] [Accepted: 01/15/2019] [Indexed: 02/07/2023]
Abstract
Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12-14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.
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Affiliation(s)
- Deepak Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - Taruna Anand
- NCVTC, ICAR-National Research Centre on Equines, Hisar 125001, Haryana, India
| | - Kennady Vijayalakshmy
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - Papori Sharma
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - Rasika Rajendran
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - Naresh L Selokar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - P S Yadav
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India
| | - Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffalo, Hisar 125001, Haryana, India.
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27
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Kim SJ, Kwon HS, Kwon DK, Koo OJ, Moon JH, Park EJ, Yum SY, Lee BC, Jang G. Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System. Cell Reprogram 2019; 21:26-36. [DOI: 10.1089/cell.2018.0036] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Affiliation(s)
- Su-Jin Kim
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Hee-Sun Kwon
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Dae-kee Kwon
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | | | - Joon-Ho Moon
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Eun-Jung Park
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Soo-Young Yum
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Byeong-Chun Lee
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
| | - Goo Jang
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
- BK21 Plus program, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Emergence Center for Food-Medicine Personalized Therapy System, Advanced Institutes of Convergence Technology, Seoul National University, Gyeonggi-do, Republic of Korea
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28
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Pieri NCG, de Souza AF, Botigelli RC, Machado LS, Ambrosio CE, Dos Santos Martins D, de Andrade AFC, Meirelles FV, Hyttel P, Bressan FF. Stem cells on regenerative and reproductive science in domestic animals. Vet Res Commun 2019; 43:7-16. [PMID: 30656543 DOI: 10.1007/s11259-019-9744-6] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2018] [Accepted: 01/09/2019] [Indexed: 12/12/2022]
Abstract
Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.
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Affiliation(s)
- Naira Caroline Godoy Pieri
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Aline Fernanda de Souza
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Ramon Cesar Botigelli
- Department of Pharmacology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil
| | - Lucas Simões Machado
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Carlos Eduardo Ambrosio
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Daniele Dos Santos Martins
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - André Furugen Cesar de Andrade
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Flavio Vieira Meirelles
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Poul Hyttel
- Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil.
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29
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Canizo JR, Vazquez Echegaray C, Klisch D, Aller JF, Paz DA, Alberio RH, Alberio R, Guberman AS. Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system. BMC Res Notes 2018; 11:509. [PMID: 30053877 PMCID: PMC6062933 DOI: 10.1186/s13104-018-3627-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Accepted: 07/20/2018] [Indexed: 02/08/2023] Open
Abstract
OBJECTIVES The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.
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Affiliation(s)
- Jesica R Canizo
- Departamento de Producción Animal, INTA EEA Balcarce, Buenos Aires, Argentina
| | - Camila Vazquez Echegaray
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.,CONICET-Universidad de Buenos Aires, Instituto de Química Biológica (IQUIBICEN), Buenos Aires, Argentina
| | - Doris Klisch
- School of Biosciences, University of Nottingham, Nottingham, LE12 5RD, UK
| | - Juan F Aller
- Departamento de Producción Animal, INTA EEA Balcarce, Buenos Aires, Argentina
| | - Dante A Paz
- Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.,Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Universidad de Buenos Aires - CONICET, Buenos Aires, Argentina
| | - Ricardo H Alberio
- Departamento de Producción Animal, INTA EEA Balcarce, Buenos Aires, Argentina.,Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Nottingham, LE12 5RD, UK
| | - Alejandra S Guberman
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina. .,CONICET-Universidad de Buenos Aires, Instituto de Química Biológica (IQUIBICEN), Buenos Aires, Argentina. .,Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
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30
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Cao H, Bian Y, Zhang F, Tang Y, Li C, Chen J, Zhang X. Functional role of Forskolin and PD166285 in the development of denuded mouse oocytes. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2017; 31:344-353. [PMID: 28920413 PMCID: PMC5838339 DOI: 10.5713/ajas.17.0441] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Revised: 07/08/2017] [Accepted: 09/04/2017] [Indexed: 12/21/2022]
Abstract
OBJECTIVE cAMP and mature promoting factor (MPF) play critical roles during the maturation of mammalian oocytes. The aim of this study was to produce the offspring from denuded oocytes (DOs) in mice by regulating cAMP and MPF. METHODS In this study, we used DOs at the germinal vesicle (GV) stage in mice and regulated levels of cAMP and MPF in DOs by adding Forskolin and PD166285 during in vitro maturation without follicle stimulating hormone and luteinizing hormone, respectively. RESULTS Combined use of 50 μM Forskolin for 3 h and 2.5 μM PD166285 for additional 21 h enhanced the developmental competence of DOs, maturation rate of DOs was 76.71%± 4.11%, blastocyst rate was 18.33%±4.44% after parthenogenetic activation (PA). The DOs could successfully be fertilized with sperm in vitro, cleavage rate was 17.02%±5.82% and blastocyst rate was 5.65%±3.10%. Besides, 2-cell in vitro fertilization embryos from DOs produced 4 normal live offspring (4/34). CONCLUSION The results confirmed that the combination of Forskolin and PD166285 can induce DOs to complete meiosis process and produce normal offspring.
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Affiliation(s)
- Hongguo Cao
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.,Anhui Provincial Laboratory of Local Animal Genetic Resources Conservation and Biobreeding, Hefei 230036, China
| | - Yani Bian
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
| | - Fei Zhang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
| | - Yunshu Tang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
| | - Caixia Li
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
| | - Jiemei Chen
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
| | - Xiaorong Zhang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.,Anhui Provincial Laboratory of Local Animal Genetic Resources Conservation and Biobreeding, Hefei 230036, China
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Rawat N, Singh MK. Induced pluripotent stem cell: A headway in reprogramming with promising approach in regenerative biology. Vet World 2017; 10:640-649. [PMID: 28717316 PMCID: PMC5499081 DOI: 10.14202/vetworld.2017.640-649] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2017] [Accepted: 04/26/2017] [Indexed: 12/17/2022] Open
Abstract
Since the embryonic stem cells have knocked the doorsteps, they have proved themselves in the field of science, research, and medicines, but the hovered restrictions confine their application in human welfare. Alternate approaches used to reprogram the cells to the pluripotent state were not up to par, but the innovation of induced pluripotent stem cells (iPSCs) paved a new hope for the researchers. Soon after the discovery, iPSCs technology is undergoing renaissance day by day, i.e., from the use of genetic material to recombinant proteins and now only chemicals are employed to convert somatic cells to iPSCs. Thus, this technique is moving straightforward and productive at an astonishing pace. Here, we provide a brief introduction to iPSCs, the mechanism and methods for their generation, their prevailing and prospective applications and the future opportunities that can be expected from them.
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Affiliation(s)
- N Rawat
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR - National Dairy Research Institute, Karnal - 132 001, Haryana, India
| | - M K Singh
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR - National Dairy Research Institute, Karnal - 132 001, Haryana, India
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32
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Characterization of the single-cell derived bovine induced pluripotent stem cells. Tissue Cell 2017; 49:521-527. [PMID: 28720304 DOI: 10.1016/j.tice.2017.05.005] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2017] [Revised: 05/19/2017] [Accepted: 05/20/2017] [Indexed: 12/12/2022]
Abstract
Single-cell derived bovine induced pluripotent stem cells (iPSCs) were generated by the introduction of piggyBac transposons with CAG promoting transcription factors (Oct3/4, Sox2, Klf4 and cMyc). In the study, the bovine iPSCs colony from single cell could passage more than 50 passages after enzymatic dissociation into single cells. These bovine iPSCs cells kept the normal karyotype and displayed dome shaped clones similar to mouse embryonic stem cells. They showed pluripotency in many ways, including their expression of pluripotency markers, such as OCT3/4, NANOG, SOX2, SSEA1, SSEA4, and AP in immunofluorescence assay, Oct4, Nanog, Sox2, Klf4 and cMyc in RT-PCR. Additionally, single-cell derived bovine iPSCs formed embryoid bodies and teratomas that all subsequently gave rise to differentiated cells from all three embryonic germ layers. The results showed that our reprogramming method could obtain high efficiency single-cell cloning bovine iPSCs, and the efficiency of single cell cloning is 40%.
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33
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Talbot NC, Sparks WO, Phillips CE, Ealy AD, Powell AM, Caperna TJ, Garrett WM, Donovan DM, Blomberg LA. Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors. Mol Reprod Dev 2017; 84:468-485. [PMID: 28332752 DOI: 10.1002/mrd.22797] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Accepted: 03/08/2017] [Indexed: 12/17/2022]
Abstract
Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.
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Affiliation(s)
- Neil C Talbot
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Wendy O Sparks
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Caitlin E Phillips
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Alan D Ealy
- Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, Virginia
| | - Anne M Powell
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Thomas J Caperna
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Wesley M Garrett
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - David M Donovan
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
| | - Le Ann Blomberg
- U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland
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34
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Yao L, Chen R, Wang P, Zhang Q, Tang H, Sun H. Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells. Am J Transl Res 2016; 8:4982-4993. [PMID: 27904699 PMCID: PMC5126341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2016] [Accepted: 10/11/2016] [Indexed: 06/06/2023]
Abstract
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.
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Affiliation(s)
- Ling Yao
- The Lab of Traditional Chinese Medicine, Chongqing Medical UniversityChongqing 400016, China
| | - Ruifang Chen
- Department of Obstetrics & Gynecology, Obstetrics & Gynecology Hospital, Fudan UniversityShanghai 200011, China
| | - Pu Wang
- Department of Neurosurgery, Huashan Hospital, Fudan UniversityShanghai 200040, China
| | - Qi Zhang
- Department of Dermatology, Huashan Hospital, Fudan UniversityShanghai 200040, China
| | - Hailiang Tang
- Department of Neurosurgery, Huashan Hospital, Fudan UniversityShanghai 200040, China
| | - Huaping Sun
- Department of Radiology, Huashan Hospital, Fudan UniversityShanghai 200040, China
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35
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Luo J, Cibelli JB. Conserved Role of bFGF and a Divergent Role of LIF for Pluripotency Maintenance and Survival in Canine Pluripotent Stem Cells. Stem Cells Dev 2016; 25:1670-1680. [DOI: 10.1089/scd.2016.0164] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Affiliation(s)
- Jiesi Luo
- Department of Animal Science, Michigan State University, East Lansing, Michigan
- Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut
| | - Jose B. Cibelli
- Department of Animal Science, Michigan State University, East Lansing, Michigan
- Department of Physiology, Michigan State University, East Lansing, Michigan
- LARCEL, Laboratorio Andaluz de Reprogramación Celular, BIONAND, Andalucía, Spain
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36
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Bai C, Li X, Gao Y, Yuan Z, Hu P, Wang H, Liu C, Guan W, Ma Y. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells. J Pineal Res 2016; 61:154-67. [PMID: 27090494 DOI: 10.1111/jpi.12334] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Accepted: 04/12/2016] [Indexed: 12/18/2022]
Abstract
Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.
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Affiliation(s)
- Chunyu Bai
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Xiangchen Li
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yuhua Gao
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- College of Wildlife Resources, Northeast Forestry University, Harbin, China
| | - Ziao Yuan
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- College of Wildlife Resources, Northeast Forestry University, Harbin, China
| | - Pengfei Hu
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China
| | - Hui Wang
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- Department of Food Science and Engineering, Liaoning Medical University, Jinzhou, China
| | - Changqing Liu
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- Department of Bioscience, Bengbu Medical College, Bengbu, China
| | - Weijun Guan
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yuehui Ma
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
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37
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Yokota S, Matsuno R, Kato H, Hashimoto H, Kinoshita M, Yokoi H, Suzuki T. Establishment of oct4:egfp transgenic and oct4:egfp /β-actin:DsRed double transgenic medaka lines. In Vitro Cell Dev Biol Anim 2016; 52:646-53. [PMID: 27067442 DOI: 10.1007/s11626-016-0020-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Accepted: 03/22/2016] [Indexed: 02/03/2023]
Abstract
As a model to examine cellular multipotency in fish, we established a medaka transgenic (Tg) Tru.oct4:egfp line carrying the green fluorescence protein (GFP) cDNA under control of the Takifugu rubripes oct4 promoter. In this Tg line, GFP could be used to examine both maternal and zygotic oct4 expression during embryogenesis. In addition, while adult Tg fish did not express GFP in any somatic cells, activation of GFP expression was initiated in regenerating fins after amputation. In vitro, some of the cell populations that migrated from fin explants expressed GFP, implying that GFP could be used to monitor oct4 expression in both embryos and in regenerating tissues in the Tru.oct4:egfp Tg line. Next, crossing with β-actin:DsRed Tg line in which all cells emit red fluorescence by expression of red fluorescent protein (RFP) under the β-actin promoter, we prepared a Tru.oct4:egfp /β-actin:DsRed double Tg line. In the double Tg line, early embryonic cells were +GFP/+RFP double positive. In vitro fin cell culture, a small number of +GFP/+RFP double positive cells could be discriminated from other -GFP/+RFP cells. Thus, when transplanted into wild-type medaka, this double Tg line can be used to trace the fate of the transplanted cells using RFP fluorescence after the loss of GFP expression.
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Affiliation(s)
- Shinpei Yokota
- Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan
| | - Rinta Matsuno
- Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan
| | - Hiroyuki Kato
- Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan
| | - Hisashi Hashimoto
- Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601, Japan
| | - Masato Kinoshita
- Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan
| | - Hayato Yokoi
- Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan
| | - Tohru Suzuki
- Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
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Zhang J, Guan J, Qi X, Ding H, Yuan H, Xie Z, Chen C, Li X, Zhang C, Huang Y. Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration. Int J Biol Sci 2016; 12:639-52. [PMID: 27194942 PMCID: PMC4870708 DOI: 10.7150/ijbs.14025] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 02/27/2016] [Indexed: 12/22/2022] Open
Abstract
The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1α in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study, we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1α and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1α. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats, DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone, leading to bone regeneration. Collectively, the results indicate that DMOG, via activation of the PI3K/Akt pathway, promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration.
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Affiliation(s)
- Jieyuan Zhang
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Junjie Guan
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Xin Qi
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Hao Ding
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Hong Yuan
- 2. Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Zongping Xie
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Chunyuan Chen
- 3. Graduate School of Nanchang University, Nanchang, Jiangxi 330031, China
| | - Xiaolin Li
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Changqing Zhang
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
| | - Yigang Huang
- 1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China
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Cravero D, Martignani E, Miretti S, Accornero P, Pauciullo A, Sharma R, Donadeu FX, Baratta M. Generation of Induced Pluripotent Stem Cells from Bovine Epithelial Cells and Partial Redirection Toward a Mammary Phenotype In Vitro. Cell Reprogram 2016; 17:211-20. [PMID: 26053520 DOI: 10.1089/cell.2014.0087] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
In contrast to adult stem cells, induced pluripotent stem cells (iPSCs) can be grown robustly in vitro and differentiated into virtually any tissue, thus providing an attractive alternative for biomedical applications. Although iPSC technology is already being used in human biomedicine, its potential in animal production has not been investigated. Herein, we investigated the potential application of iPSCs in dairy production by generating bovine iPSCs and establishing their ability to generate mammary epithelial tissue. iPSCs were derived by retrovirus-mediated expression of murine Oct4, Sox2, Klf4, and c-Myc in mammary epithelium and dermal fibroblasts. The resulting reprogrammed cells stained positive for alkaline phosphatase and showed renewed expression of pluripotency genes, including Lin28, Rex1, Oct4, Sox2, and Nanog. In addition, injection of epithelial- or fibroblast-derived reprogrammed cells into nonobese diabetic (NOD/NOD) mice resulted in the formation of teratomas containing differentiated derivatives of the three germ layers, including cartilage, membranous ossification, stratified squamous epithelial tissue, hair follicles, neural pinwheels, and different types of glandular tissue. Finally, mammary epithelium-derived iPSCs could be induced to differentiate back to a mammary phenotype characterized by epithelial cells expressing cytokeratin 14 (CK14), CK18, and smooth muscle actin (SMA) as a result of treatment with 10 nM progesterone. This study reports for the first time the generation of iPSCs from bovine epithelial cells and demonstrates the potential of using iPSCs technology for generating bovine mammary tissue in vitro.
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Affiliation(s)
- Diego Cravero
- 1 Department of Veterinary Science, University of Torino , 10095 Grugliasco (TO), Italy
| | - Eugenio Martignani
- 1 Department of Veterinary Science, University of Torino , 10095 Grugliasco (TO), Italy
| | - Silvia Miretti
- 1 Department of Veterinary Science, University of Torino , 10095 Grugliasco (TO), Italy
| | - Paulo Accornero
- 1 Department of Veterinary Science, University of Torino , 10095 Grugliasco (TO), Italy
| | - Alfredo Pauciullo
- 2 Department Agricultural, Forest and Food Sciences, University of Torino , Grugliasco (TO), Italy
| | - Ruchi Sharma
- 3 The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh , Midlothian, United Kingdom
| | - Francesco Xavier Donadeu
- 3 The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh , Midlothian, United Kingdom
| | - Mario Baratta
- 1 Department of Veterinary Science, University of Torino , 10095 Grugliasco (TO), Italy
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40
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Ogorevc J, Orehek S, Dovč P. Cellular reprogramming in farm animals: an overview of iPSC generation in the mammalian farm animal species. J Anim Sci Biotechnol 2016; 7:10. [PMID: 26900466 PMCID: PMC4761155 DOI: 10.1186/s40104-016-0070-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2015] [Accepted: 02/11/2016] [Indexed: 12/19/2022] Open
Abstract
Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism’s body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven’t received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format.
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Affiliation(s)
- J Ogorevc
- Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia
| | - S Orehek
- Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia
| | - P Dovč
- Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia
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41
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Pluripotent stem cells and livestock genetic engineering. Transgenic Res 2016; 25:289-306. [PMID: 26894405 DOI: 10.1007/s11248-016-9929-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2015] [Accepted: 01/06/2016] [Indexed: 01/12/2023]
Abstract
The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.
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Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells. Stem Cells Int 2016; 2016:5127984. [PMID: 26880968 PMCID: PMC4736581 DOI: 10.1155/2016/5127984] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Revised: 10/25/2015] [Accepted: 10/29/2015] [Indexed: 11/17/2022] Open
Abstract
Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential.
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Abstract
This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably stem from an inability to maintain the stability of the endogenous gene networks necessary to maintain pluripotency. Based on work in humans and rodents, achievement of full pluripotency will likely require fine adjustments in the growth factors and signaling inhibitors provided to the cells. Finally, we discuss the future utility of these cells for biomedical and agricultural purposes.
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Affiliation(s)
- Toshihiko Ezashi
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - Ye Yuan
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
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Nong W, Xie TS, Li LY, Lu AG, Mo J, Gou YF, Lan G, Jiang H, Len J, Li MM, Jiang QY, Huang B. Qualitative Analyses of Protein Phosphorylation in Bovine Pluripotent Stem Cells Generated from Embryonic Fibroblasts. Reprod Domest Anim 2015; 50:989-98. [DOI: 10.1111/rda.12619] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2015] [Accepted: 09/07/2015] [Indexed: 12/19/2022]
Affiliation(s)
- W Nong
- College of Animal Science and Technology; Guangxi University; Nanning China
- Guangxi University of Chinese Medicine; Nanning China
| | - TS Xie
- College of Animal Science and Technology; Guangxi University; Nanning China
- Nanning Languang Biotechnology Inc.; Nanning China
| | - LY Li
- College of Animal Science and Technology; Guangxi University; Nanning China
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources; Guangxi University; Nanning China
| | - AG Lu
- College of Animal Science and Technology; Guangxi University; Nanning China
- Guangxi Analysis and Testing Center; Nanning China
| | - J Mo
- Guangxi Analysis and Testing Center; Nanning China
| | - YF Gou
- College of Animal Science and Technology; Guangxi University; Nanning China
| | - G Lan
- College of Animal Science and Technology; Guangxi University; Nanning China
| | - H Jiang
- College of Animal Science and Technology; Guangxi University; Nanning China
| | - J Len
- Guangxi University of Chinese Medicine; Nanning China
| | - MM Li
- College of Animal Science and Technology; Guangxi University; Nanning China
| | - QY Jiang
- College of Animal Science and Technology; Guangxi University; Nanning China
| | - B Huang
- College of Animal Science and Technology; Guangxi University; Nanning China
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources; Guangxi University; Nanning China
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Talluri TR, Kumar D, Glage S, Garrels W, Ivics Z, Debowski K, Behr R, Niemann H, Kues WA. Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming. Cell Reprogram 2015; 17:131-40. [PMID: 25826726 DOI: 10.1089/cell.2014.0080] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line-competent bovine iPSCs and will facilitate genetic modification of the bovine genome.
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Affiliation(s)
- Thirumala R Talluri
- 1 Institut für Nutztiergenetik, Friedrich-Loeffler-Institut , Mariensee, 31535 Neustadt, Germany
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Kim D, Park S, Jung YG, Roh S. In vitro culture of stem-like cells derived from somatic cell nuclear transfer bovine embryos of the Korean beef cattle species, HanWoo. Reprod Fertil Dev 2015; 28:RD14071. [PMID: 25966803 DOI: 10.1071/rd14071] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2014] [Accepted: 04/12/2015] [Indexed: 12/27/2022] Open
Abstract
We established and maintained somatic cell nuclear transfer embryo-derived stem-like cells (SCNT-eSLCs) from the traditional Korean beef cattle species, HanWoo (Bos taurus coreanae). Each SCNT blastocyst was placed individually on a feeder layer with culture medium containing three inhibitors of differentiation (3i). Primary colonies formed after 2-3 days of culture and the intact colonies were passaged every 5-6 days. The cells in each colony showed embryonic stem cell-like morphologies with a distinct boundary and were positive to alkaline phosphatase staining. Immunofluorescence and reverse transcription-polymerase chain reaction analyses also confirmed that these colonies expressed pluripotent markers. The colonies were maintained over 50 passages for more than 270 days. The cells showed normal karyotypes consisting of 60 chromosomes at Passage 50. Embryoid bodies were formed by suspension culture to analyse in vitro differentiation capability. Marker genes representing the differentiation into three germ layers were expressed. Typical embryonal carcinoma was generated after injecting cells under the testis capsule of nude mice, suggesting that the cultured cells may also have the potential of in vivo differentiation. In conclusion, we generated eSLCs from SCNT bovine embryos, using a 3i system that sustained stemness, normal karyotype and pluripotency, which was confirmed by in vitro and in vivo differentiation.
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Kumar D, Talluri TR, Anand T, Kues WA. Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals. World J Stem Cells 2015; 7:315-328. [PMID: 25815117 PMCID: PMC4369489 DOI: 10.4252/wjsc.v7.i2.315] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 08/19/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.
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Whitworth DJ, Banks TA. Stem cell therapies for treating osteoarthritis: prescient or premature? Vet J 2014; 202:416-24. [PMID: 25457267 DOI: 10.1016/j.tvjl.2014.09.024] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2013] [Revised: 09/22/2014] [Accepted: 09/25/2014] [Indexed: 01/23/2023]
Abstract
There has been unprecedented interest in recent years in the use of stem cells as therapy for an array of diseases in companion animals. Stem cells have already been deployed therapeutically in a number of clinical settings, in particular the use of mesenchymal stem cells to treat osteoarthritis in horses and dogs. However, an assessment of the scientific literature highlights a marked disparity between the purported benefits of stem cell therapies and their proven abilities as defined by rigorously controlled scientific studies. Although preliminary data generated from clinical trials in human patients are encouraging, therapies currently available to treat animals are supported by very limited clinical evidence, and the commercialisation of these treatments may be premature. This review introduces the three main types of stem cells relevant to veterinary applications, namely, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells, and draws together research findings from in vitro and in vivo studies to give an overview of current stem cell therapies for the treatment of osteoarthritis in animals. Recent advances in tissue engineering, which is proposed as the future direction of stem cell-based therapy for osteoarthritis, are also discussed.
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Affiliation(s)
- Deanne J Whitworth
- School of Veterinary Science, University of Queensland, Gatton, Queensland 4343, Australia.
| | - Tania A Banks
- School of Veterinary Science, University of Queensland, Gatton, Queensland 4343, Australia
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Koh S, Piedrahita JA. From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals. Theriogenology 2014; 81:103-11. [PMID: 24274415 DOI: 10.1016/j.theriogenology.2013.09.009] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2013] [Revised: 09/05/2013] [Accepted: 09/05/2013] [Indexed: 01/10/2023]
Abstract
Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as "embryonic stem-like" cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs.
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Affiliation(s)
- Sehwon Koh
- Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, USA; Genomics Program, North Carolina State University, Raleigh, North Carolina, USA
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50
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Yu P, Lu Y, Jordan BJ, Liu Y, Yang JY, Hutcheson JM, Ethridge CL, Mumaw JL, Kinder HA, Beckstead RB, Stice SL, West FD. Nonviral minicircle generation of induced pluripotent stem cells compatible with production of chimeric chickens. Cell Reprogram 2014; 16:366-78. [PMID: 25084370 DOI: 10.1089/cell.2014.0028] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such as meat and egg production or even disease resistance. However, these cell types have significant limitations because they are hard to culture long term while maintaining developmental plasticity. Induced pluripotent stem cells (iPSCs) are a novel class of stem cells that have proven to be robust, leading to the successful development of transgenic mice, rats, quail, and pigs and may potentially overcome the limitations of previous pluripotent stem cell systems in chickens. In this study we generated chicken (c) iPSCs from fibroblast cells for the first time using a nonviral minicircle reprogramming approach. ciPSCs demonstrated stem cell morphology and expressed key stem cell markers, including alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1. These cells were capable of rapid growth and expressed high levels of telomerase. Late-passage ciPSCs transplanted into stage X embryos were successfully incorporated into tissues of all three germ layers, and the gonads demonstrated significant cellular plasticity. These cells provide an exciting new tool to create transgenic chickens with broad implications for agricultural and transgenic animal fields at large.
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Affiliation(s)
- Ping Yu
- 1 Regenerative Bioscience Center, University of Georgia , Athens, GA, 30602
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