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1
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Bao Q, Tay NL, Lim CY, Chua DHH, Kee SK, Choolani M, Loh YH, Ng SC, Chai C. Integration-free induced pluripotent stem cells from three endangered Southeast Asian non-human primate species. Sci Rep 2024; 14:2391. [PMID: 38287040 PMCID: PMC10825216 DOI: 10.1038/s41598-023-50510-9] [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: 07/24/2023] [Accepted: 12/20/2023] [Indexed: 01/31/2024] Open
Abstract
Advanced molecular and cellular technologies provide promising tools for wildlife and biodiversity conservation. Induced pluripotent stem cell (iPSC) technology offers an easily accessible and infinite source of pluripotent stem cells, and have been derived from many threatened wildlife species. This paper describes the first successful integration-free reprogramming of adult somatic cells to iPSCs, and their differentiation, from three endangered Southeast Asian primates: the Celebes Crested Macaque (Macaca nigra), the Lar Gibbon (Hylobates lar), and the Siamang (Symphalangus syndactylus). iPSCs were also generated from the Proboscis Monkey (Nasalis larvatus). Differences in mechanisms could elicit new discoveries regarding primate evolution and development. iPSCs from endangered species provides a safety net in conservation efforts and allows for sustainable sampling for research and conservation, all while providing a platform for the development of further in vitro models of disease.
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Affiliation(s)
- Qiuye Bao
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore
| | - Nicole Liling Tay
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore
- Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore
| | - Christina Yingyan Lim
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore
| | | | - Su Keyau Kee
- Cytogenetics Laboratory, Department of Pathology, Singapore General Hospital, 20 College Road, Singapore, 169856, Singapore
| | - Mahesh Choolani
- Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore
| | - Yuin-Han Loh
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117593, Singapore
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore
| | - Soon Chye Ng
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore.
- Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore.
- Sincere Healthcare Group, 8 Sinaran Drive, Singapore, 307470, Singapore.
| | - Chou Chai
- Institute of Molecular and Cell Biology-Endangered Species Conservation By Assisted Reproduction (IMCB-ESCAR) Joint Laboratory, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Singapore, 138673, Singapore
- Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore, 308232, Singapore
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2
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Swegen A, Appeltant R, Williams SA. Cloning in action: can embryo splitting, induced pluripotency and somatic cell nuclear transfer contribute to endangered species conservation? Biol Rev Camb Philos Soc 2023; 98:1225-1249. [PMID: 37016502 DOI: 10.1111/brv.12951] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Revised: 03/11/2023] [Accepted: 03/13/2023] [Indexed: 04/06/2023]
Abstract
The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.
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Affiliation(s)
- Aleona Swegen
- Nuffield Department of Women's and Reproductive Health, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
- Priority Research Centre for Reproductive Science, University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia
| | - Ruth Appeltant
- Nuffield Department of Women's and Reproductive Health, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
- Gamete Research Centre, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium
| | - Suzannah A Williams
- Nuffield Department of Women's and Reproductive Health, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
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3
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Zhao L, Long C, Zhao G, Su J, Ren J, Sun W, Wang Z, Zhang J, Liu M, Hao C, Li H, Cao G, Bao S, Zuo Y, Li X. Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis. J Cell Mol Med 2022; 26:4792-4804. [PMID: 35971640 PMCID: PMC9465183 DOI: 10.1111/jcmm.17505] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 06/05/2022] [Accepted: 07/11/2022] [Indexed: 11/28/2022] Open
Abstract
Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single‐cell RNA sequencing (scRNA‐seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA‐seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8‐cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2‐cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.
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Affiliation(s)
- Lixia Zhao
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
| | - Chunshen Long
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Gaoping Zhao
- Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
| | - Jie Su
- Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China.,College of Veterinary Medicine, Key Laboratory of Basic Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, China
| | - Jie Ren
- Beijing Advanced Innovation Center for Genomics, College of Life Sciences, Peking University, Beijing, China
| | - Wei Sun
- Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
| | - Zixin Wang
- Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
| | - Jia Zhang
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Moning Liu
- College of Veterinary Medicine, Key Laboratory of Basic Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, China
| | - Chunxia Hao
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Hanshuang Li
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Guifang Cao
- College of Veterinary Medicine, Key Laboratory of Basic Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, China
| | - Siqin Bao
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yongchun Zuo
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Xihe Li
- The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
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4
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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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5
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Gavin-Plagne L, Perold F, Osteil P, Voisin S, Moreira SC, Combourieu Q, Saïdou V, Mure M, Louis G, Baudot A, Buff S, Joly T, Afanassieff M. Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells. Int J Mol Sci 2020; 21:ijms21197285. [PMID: 33023104 PMCID: PMC7582889 DOI: 10.3390/ijms21197285] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 09/18/2020] [Accepted: 09/28/2020] [Indexed: 12/11/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0-5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.
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Affiliation(s)
- Lucie Gavin-Plagne
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
- Univ Lyon, Université Claude Bernard Lyon 1, VetAgro Sup, UPSP ICE 2016.A104, F-69280 Marcy l’Etoile, France; (S.B.); (T.J.)
| | - Florence Perold
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Pierre Osteil
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Sophie Voisin
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Synara Cristina Moreira
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Quitterie Combourieu
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Véronique Saïdou
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Magali Mure
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
| | - Gérard Louis
- Univ Paris, Université Descartes Paris V, LVTS, Inserm UMRS 1148, F-75018 Paris, France; (G.L.); (A.B.)
| | - Anne Baudot
- Univ Paris, Université Descartes Paris V, LVTS, Inserm UMRS 1148, F-75018 Paris, France; (G.L.); (A.B.)
| | - Samuel Buff
- Univ Lyon, Université Claude Bernard Lyon 1, VetAgro Sup, UPSP ICE 2016.A104, F-69280 Marcy l’Etoile, France; (S.B.); (T.J.)
| | - Thierry Joly
- Univ Lyon, Université Claude Bernard Lyon 1, VetAgro Sup, UPSP ICE 2016.A104, F-69280 Marcy l’Etoile, France; (S.B.); (T.J.)
- Univ Lyon, Université Claude Bernard Lyon 1, ISARA-Lyon, UPSP ICE 2016.A104, F-69007 Lyon, France
| | - Marielle Afanassieff
- Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U 1208, USC 1361, F-69500 Bron, France; (L.G.-P.); (F.P.); (P.O.); (S.V.); (S.C.M.); (Q.C.); (V.S.); (M.M.)
- Correspondence: ; Tel.: +33-472-913-458
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6
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Pan S, Chen YC, Zhao N, Feng X, Yang DD, Wang Y, Jin ZB. A new subset of small stem cells in bovine bone marrow stromal cell populations. J Cell Biochem 2019; 120:13881-13892. [PMID: 30983000 DOI: 10.1002/jcb.28661] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Revised: 12/21/2018] [Accepted: 01/07/2019] [Indexed: 01/23/2023]
Abstract
Bone marrow stromal cells (BMSCs) are a unique population of multipotent cells that exhibit pluripotent properties to a certain extent and are significantly heterogeneous in terms of the cell population. We isolate a small cell subpopulation from bovine BMSCs, bovine small stem cells (bSSCs), and herein characterize their properties. The bSSCs are smaller in size and express nuclear Oct-4 and other pluripotency markers. In addition, when cultured in suspension conditions, bSSCs form three-dimensional spheres and display a strong capability for self-renewal and differentiation into cells from three germ layers. Notably, bSSCs display neural features with Sox1 and Pax6 expression. Using bSSCs as donor nuclear cells for somatic cell nuclear transfer, we further demonstrate that the developmental potential of cloned embryos in vitro is significantly increased. Our study identifies a new bovine bone marrow stromal cell-derived stem cell subtype that could have broad importance for developmental biology as well as great potential for regenerative medicine.
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Affiliation(s)
- Shaohui Pan
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
| | - Yu-Chen Chen
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
| | - Ning Zhao
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
| | - Xiang Feng
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
| | - Dan-Dan Yang
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
| | - Yongshen Wang
- Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, China
| | - Zi-Bing Jin
- Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China.,State Key Laboratory of Ophthalmology, Optometry and Visual Sciences, Wenzhou, China
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7
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Saadeldin IM, Abdel-Aziz Swelum A, Alzahrani FA, Alowaimer AN. The current perspectives of dromedary camel stem cells research. Int J Vet Sci Med 2018; 6:S27-S30. [PMID: 30761317 PMCID: PMC6161867 DOI: 10.1016/j.ijvsm.2018.01.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Accepted: 01/06/2018] [Indexed: 12/17/2022] Open
Abstract
Camels have cultural value in the Arab society and are considered one of the most important animals in the Arabian Peninsula and arid environments, due to the distinct characteristics of their meat and milk. Moreover, there is a great interest in camel racing and beauty shows. Therefore, treatment of elite animals, increasing the number of camels as well as genetic improvement is an essential demand. Because there are unique camels for milk production, meat, or in racing, the need to propagate genetically superior camels is urgent. Recent biotechnological approaches such as stem cells hold great promise for biomedical research, genetic engineering, and as a model for studying early mammalian developmental biology. Establishment of stem cells lines from camels would tremendously facilitate regenerative medicine for genetically superior camels, permit the gene targeting of the camel genome and the generation of genetically modified animal and be a mean for genome conservation for the elite breeds. In this mini-review, we show the current research, future horizons and potential applications for camel stem cells.
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Affiliation(s)
- Islam M Saadeldin
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, 11451 Riyadh, Saudi Arabia.,Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt
| | - Ayman Abdel-Aziz Swelum
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, 11451 Riyadh, Saudi Arabia.,Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt
| | - Faisal A Alzahrani
- Department of Biological Sciences, Rabigh College of Science and Arts, King Abdulaziz University, Rabigh Branch, Rabigh 21911, Saudi Arabia
| | - Abdullah N Alowaimer
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, 11451 Riyadh, Saudi Arabia
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8
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Song H, Li H, Huang M, Xu D, Wang Z, Wang F. Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells. Cell Reprogram 2016; 18:37-47. [PMID: 26836033 DOI: 10.1089/cell.2015.0035] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.
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Affiliation(s)
- Hui Song
- 1 Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University , Nanjing, 210095, P.R. China .,2 Department of Medical Genetic and Cell Biology, Ningxia Medical University , Yinchuan, 750004, China
| | - Hui Li
- 1 Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University , Nanjing, 210095, P.R. China .,2 Department of Medical Genetic and Cell Biology, Ningxia Medical University , Yinchuan, 750004, China
| | - Mingrui Huang
- 1 Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University , Nanjing, 210095, P.R. China
| | - Dan Xu
- 3 Stanford University School of Medicine , Stanford, CA, 94305
| | - Ziyu Wang
- 1 Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University , Nanjing, 210095, P.R. China
| | - Feng Wang
- 1 Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University , Nanjing, 210095, P.R. China
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9
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Olivera R, Moro LN, Jordan R, Luzzani C, Miriuka S, Radrizzani M, Donadeu FX, Vichera G. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors. PLoS One 2016; 11:e0164049. [PMID: 27732616 PMCID: PMC5061425 DOI: 10.1371/journal.pone.0164049] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2016] [Accepted: 09/19/2016] [Indexed: 02/06/2023] Open
Abstract
The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.
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Affiliation(s)
| | - Lucia Natalia Moro
- Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina
| | | | - Carlos Luzzani
- Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina
| | - Santiago Miriuka
- Laboratory of Biology of Cell Development, LIAN-Unit associated with CONICET, FLENI, Belen de Escobar, Buenos Aires, Argentina
| | - Martin Radrizzani
- Laboratory of Neruogenetic and Molecular Cytogentic, School of Sciences, National University of San Martin, CONICET, Buenos Aires, Argentina
| | - F. Xavier Donadeu
- The Roslin Institute and Royal School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom
- Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, United Kingdom
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10
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Du X, Feng T, Yu D, Wu Y, Zou H, Ma S, Feng C, Huang Y, Ouyang H, Hu X, Pan D, Li N, Wu S. Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors. Stem Cells 2016; 33:3228-38. [PMID: 26138940 DOI: 10.1002/stem.2089] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 05/23/2015] [Accepted: 06/15/2015] [Indexed: 12/11/2022]
Abstract
To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets.
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Affiliation(s)
- Xuguang Du
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Tao Feng
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Dawei Yu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Yuanyuan Wu
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA
| | - Huiying Zou
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Shuangyu Ma
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Chong Feng
- Department of Gene and Cell Engineering, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China
| | - Yongye Huang
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, People's Republic of China
| | - Hongsheng Ouyang
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, People's Republic of China
| | - Xiaoxiang Hu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Dengke Pan
- Department of Gene and Cell Engineering, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China
| | - Ning Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Sen Wu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
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11
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Kim E, Zheng Z, Jeon Y, Jin YX, Hwang SU, Cai L, Lee CK, Kim NH, Hyun SH. An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase. PLoS One 2016; 11:e0160289. [PMID: 27472781 PMCID: PMC4966966 DOI: 10.1371/journal.pone.0160289] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Accepted: 07/15/2016] [Indexed: 12/29/2022] Open
Abstract
Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.
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Affiliation(s)
- Eunhye Kim
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
- Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Zhong Zheng
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Yubyeol Jeon
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Yong-Xun Jin
- Department of Animal Sciences, Agriculture, Life, & Environmental Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Seon-Ung Hwang
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
- Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Lian Cai
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Chang-Kyu Lee
- Department of Agricultural Biotechnology, Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea
| | - Nam-Hyung Kim
- Department of Animal Sciences, Agriculture, Life, & Environmental Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Sang-Hwan Hyun
- Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
- Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
- * E-mail:
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12
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Derivation and application of pluripotent stem cells for regenerative medicine. SCIENCE CHINA-LIFE SCIENCES 2016; 59:576-83. [DOI: 10.1007/s11427-016-5066-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 04/20/2016] [Indexed: 01/21/2023]
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13
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Park KM, Lee J, Hussein KH, Hong SH, Yang SR, Lee E, Woo HM. Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer. J Vet Med Sci 2016; 78:709-13. [PMID: 26725870 PMCID: PMC4873867 DOI: 10.1292/jvms.15-0363] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for
the development of genetically engineered pig models, because they can be expanded without
senescence and have the potential for multiple gene manipulation. They are also useful
cell sources for disease modeling and treatment. However, the generation of transgenic
porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has
not yet been reported. We report here the generation of liver-specific oncogenes
(TGF-α/c-Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They
expressed stem cell characteristics and were differentiated into hepatocyte-like cells
that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells
successfully developed blastocysts in vitro. As an initial approach
toward porcine transgenic iPS cell generation and their developmental competence after NT,
this study provides foundations for the efficient generation of genetically modified
porcine iPS cells and animal models.
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Affiliation(s)
- Kyung-Mee Park
- Stem Cell Institute-KNU, Kangwon National University, Chuncheon, 200-701, Korea
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14
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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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15
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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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16
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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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17
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Prescott HMA, Manning C, Gardner A, Ritchie WA, Pizzi R, Girling S, Valentine I, Wang C, Jahoda CAB. Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells. PLoS One 2015; 10:e0138840. [PMID: 26398672 PMCID: PMC4580591 DOI: 10.1371/journal.pone.0138840] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 09/03/2015] [Indexed: 01/01/2023] Open
Abstract
Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.
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Affiliation(s)
- Hilary M. A. Prescott
- Durham University, School of Biological and Biomedical Sciences, Durham, DH1 3LE, United Kingdom
| | - Craig Manning
- Durham University, School of Biological and Biomedical Sciences, Durham, DH1 3LE, United Kingdom
| | - Aaron Gardner
- Durham University, School of Biological and Biomedical Sciences, Durham, DH1 3LE, United Kingdom
| | - William A. Ritchie
- Roslin Embryology Ltd., 21 St Germains Terrace, Macmerry, East Lothian, EH33 1QB, United Kingdom
| | - Romain Pizzi
- Royal Zoological Society of Scotland, Corstorphine Road, Edinburgh, EH13 6TS, United Kingdom
| | - Simon Girling
- Royal Zoological Society of Scotland, Corstorphine Road, Edinburgh, EH13 6TS, United Kingdom
| | - Iain Valentine
- Royal Zoological Society of Scotland, Corstorphine Road, Edinburgh, EH13 6TS, United Kingdom
| | - Chengdong Wang
- China Conservation and Research Centre for Giant Panda (CCRCGP), Shi Qiao Village, Qing Chenshan Town, DuJiangYan City, SiChuan Province, 611844, China
| | - Colin A. B. Jahoda
- Durham University, School of Biological and Biomedical Sciences, Durham, DH1 3LE, United Kingdom
- * E-mail:
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Abstract
Nuclear transfer (NT) technique provides a powerful experimental tool to study the mechanisms of reprogramming processes and to derive NT-embryonic stem (ntES) cells from living or frozen animals. The Piezo-driven direct microinjection NT method has proved to be a valid technique to clone mice and other species. In addition, this method has been broadly used as a versatile tool for many fields of mouse micromanipulation. This chapter describes the "one step method" protocol of nuclear transfer in mouse, which combines injection of a donor cell nucleus and enucleation of MII metaphase in a single manipulation procedure. This protocol describes the isolation and collection of oocytes, treatment of donor cells, visualization of spindle-chromosomal complex, direct injection and enucleation, activation of reconstructed embryos and their in vitro culture and transfer into pseudopregnant mice.
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Affiliation(s)
- Vincent Brochard
- INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, 78350, France
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19
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German SD, Campbell KHS, Thornton E, McLachlan G, Sweetman D, Alberio R. Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer. Cell Reprogram 2014; 17:19-27. [PMID: 25513856 DOI: 10.1089/cell.2014.0071] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.
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Affiliation(s)
- Sergio D German
- 1 Division of Animal Sciences, School of Biosciences, University of Nottingham , Loughborough, LE12 5RD, United Kingdom
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Gu Q, Hao J, Hai T, Wang J, Jia Y, Kong Q, Wang J, Feng C, Xue B, Xie B, Liu S, Li J, He Y, Sun J, Liu L, Wang L, Liu Z, Zhou Q. Efficient generation of mouse ESCs-like pig induced pluripotent stem cells. Protein Cell 2014; 5:338-42. [PMID: 24671760 PMCID: PMC3996154 DOI: 10.1007/s13238-014-0043-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Affiliation(s)
- Qi Gu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
- Intelligent Polymer Research Institute, ARC Center of Excellence for Electromaterials Science, AIIM Facility, University of Wollongong, Wollongong, NSW 2522 Australia
| | - Jie Hao
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Tang Hai
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Jianyu Wang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Yundan Jia
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Qingran Kong
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Juan Wang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Chunjing Feng
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Binghua Xue
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Bingteng Xie
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Shichao Liu
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Jinyu Li
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Yilong He
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Jialu Sun
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Lei Liu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Liu Wang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
| | - Zhonghua Liu
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030 China
| | - Qi Zhou
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China
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Sanal MG. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer. SAGE Open Med 2014; 2:2050312114550375. [PMID: 26770740 PMCID: PMC4607203 DOI: 10.1177/2050312114550375] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 08/06/2014] [Indexed: 02/06/2023] Open
Abstract
Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC), we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT) made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient's somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT) combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system) this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is the question of reproductive freedom and existence.
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Wang J, Gu Q, Hao J, Jia Y, Xue B, Jin H, Ma J, Wei R, Hai T, Kong Q, Bou G, Xia P, Zhou Q, Wang L, Liu Z. Tbx3 and Nr5α2 play important roles in pig pluripotent stem cells. Stem Cell Rev Rep 2014; 9:700-8. [PMID: 23625189 DOI: 10.1007/s12015-013-9439-2] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Pigs are valuable animal models in pre-clinical research due to their anatomical and similarity to human-beings. Little is known about porcine embryonic development and porcine pluripotent stem cells. Recently, porcine-induced pluripotent stem cells (piPSCs) have been generated with Oct4 (Pou5f1), Sox2, Klf4 and c-Myc (termed OSKM, 4 F). Here, we found two other factors (Tbx3 and Nr5α2, termed TN), with important roles in piPSCs induction. They could improve the generation of piPSCs by supplementing these two factors on the basis of OSKM (OSKMTN, 6 F) orientated to mouse ESCs-like. Surprisingly, Nr5α2 alone could induce piPSCs formation in the presence or absence of c-Myc. These results suggested that Tbx3 and Nr5α2 may have vital roles in Sus scrofa and proposed new insights into pig pluripotent stem cells.
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Affiliation(s)
- Jianyu Wang
- College of Life Science, Northeast Agricultural University of China, Harbin, 150030, People's Republic China
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23
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Xie B, Wang J, Liu S, Wang J, Xue B, Li J, Wei R, Zhao Y, Liu Z. Positive correlation between the efficiency of induced pluripotent stem cells and the development rate of nuclear transfer embryos when the same porcine embryonic fibroblast lines are used as donor cells. Cell Reprogram 2014; 16:206-14. [PMID: 24738969 DOI: 10.1089/cell.2013.0080] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) and nuclear transfer (NT) are two of the primary routes to reprogram differentiated cells back to the pluripotent state. However, it is still unknown whether there is any correlation between the reprogramming efficiency of iPSCs and NT if the same donor cells are employed. In this study, six porcine embryonic fibroblast (PEF) lines from Landrace (L1, L6, L9) or Congjiang local pigs (C4, C5, C6) were used for iPSC induction and NT. Furthermore, the resultant iPSCs from four PEF lines (L1, L6, C4, and C5) were used for NT (iPSC-NT), and the expression of exogenous genes was detected in iPSC-NT embryos by real-time PCR. The results showed that the efficiency of iPSC lines established from different PEF lines were significantly different. When the same PEF lines were used as donor cells for NT, the blastocysts rates were also different among different PEF lines and positively related with iPSCs induction efficiency. When the iPSCs were used as donor cells for NT, compared with the source PEFs, the blastocysts rates were significantly decreased. Real-time PCR results indicated that exogenous genes (Oct4, c-Myc) continued to be expressed in iPSC-NT embryos. In summary, our results demonstrate that there was a positive correlation between iPSCs and NT reprogramming efficiency, although the mechanism of these two routes is different. This may provide a new method to select the appropriate donor cells for inducing iPSCs.
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Affiliation(s)
- Bingteng Xie
- 1 College of Life Science, Northeast Agricultural University of China , Harbin, 150030, China
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24
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Yuan Y, Lee K, Park KW, Spate LD, Prather RS, Wells KD, Roberts RM. Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos. Cell Cycle 2014; 13:1265-76. [PMID: 24621508 DOI: 10.4161/cc.28176] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.
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Affiliation(s)
- Ye Yuan
- Division of Animal Sciences and Bond Life Sciences Center; University of Missouri; Columbia, MO USA
| | - Kiho Lee
- Division of Animal Sciences; University of Missouri; Columbia, MO USA
| | - Kwang-Wook Park
- Division of Animal Sciences; University of Missouri; Columbia, MO USA; Department of Animal Science and Technology; Sunchon National University; Suncheon, Jeonnam, Korea
| | - Lee D Spate
- Division of Animal Sciences; University of Missouri; Columbia, MO USA
| | - Randall S Prather
- Division of Animal Sciences; University of Missouri; Columbia, MO USA
| | - Kevin D Wells
- Division of Animal Sciences; University of Missouri; Columbia, MO USA
| | - R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center; University of Missouri; Columbia, MO USA
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25
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Song H, Li H, Huang M, Xu D, Gu C, Wang Z, Dong F, Wang F. Induced pluripotent stem cells from goat fibroblasts. Mol Reprod Dev 2013; 80:1009-17. [PMID: 24123501 DOI: 10.1002/mrd.22266] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2013] [Accepted: 09/27/2013] [Indexed: 01/06/2023]
Abstract
Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo.
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Affiliation(s)
- Hui Song
- Jiangsu Engineering Technology Research Center of Meat Sheep & Goat Industry, Nanjing Agricultural University, Nanjing, People's Republic of China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, People's Republic of China
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26
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Fan N, Chen J, Shang Z, Dou H, Ji G, Zou Q, Wu L, He L, Wang F, Liu K, Liu N, Han J, Zhou Q, Pan D, Yang D, Zhao B, Ouyang Z, Liu Z, Zhao Y, Lin L, Zhong C, Wang Q, Wang S, Xu Y, Luan J, Liang Y, Yang Z, Li J, Lu C, Vajta G, Li Z, Ouyang H, Wang H, Wang Y, Yang Y, Liu Z, Wei H, Luan Z, Esteban MA, Deng H, Yang H, Pei D, Li N, Pei G, Liu L, Du Y, Xiao L, Lai L. Piglets cloned from induced pluripotent stem cells. Cell Res 2013; 23:162-166. [PMID: 23247628 PMCID: PMC3541650 DOI: 10.1038/cr.2012.176] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Affiliation(s)
- Nana Fan
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
- School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China
| | - Jijun Chen
- College of Animal Sciences, Stem Cell and Developmental Biology Research Center, Zhejiang University, Hangzhou, Zhejiang 310058, China
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | | | - Hongwei Dou
- BGI Ark Biotechnology Co., Ltd., Shenzhen, Guangdong 518083, China
| | - Guangzhen Ji
- Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Qingjian Zou
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
- School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China
| | - Lu Wu
- College of Animal Sciences, Stem Cell and Developmental Biology Research Center, Zhejiang University, Hangzhou, Zhejiang 310058, China
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Lixiazi He
- College of Animal Sciences, Stem Cell and Developmental Biology Research Center, Zhejiang University, Hangzhou, Zhejiang 310058, China
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Fang Wang
- Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Kai Liu
- Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Na Liu
- Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Jianyong Han
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Qi Zhou
- Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Dengke Pan
- Department of Gene and Cell Engineering, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
| | - Dongshan Yang
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Bentian Zhao
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Zhen Ouyang
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Zhaoming Liu
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Yu Zhao
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Lin Lin
- BGI Ark Biotechnology Co., Ltd., Shenzhen, Guangdong 518083, China
| | | | | | - Shouqi Wang
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Ying Xu
- BGI Ark Biotechnology Co., Ltd., Shenzhen, Guangdong 518083, China
| | - Jing Luan
- BGI Ark Biotechnology Co., Ltd., Shenzhen, Guangdong 518083, China
| | - Yu Liang
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China
| | | | - Jing Li
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China
| | - Chunxia Lu
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China
| | - Gábor Vajta
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China
- Central Queensland University, Bruce Highway, Rockhampton, QLD 4702, Australia
| | - Ziyi Li
- College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Hongsheng Ouyang
- College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Huayan Wang
- Department of Animal Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Yong Wang
- Department of Laboratory Animal Sciences, College of Basic medicine, Third Military Medical University, Chongqing 400038, China
| | - Yang Yang
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing 100101, China
- Center for Life Sciences, Peking University, Beijing 100101, China
| | - Zhonghua Liu
- Life Science College, North-east Agricultural University, Harbin, Heilongjiang 150030, China
| | - Hong Wei
- Department of Laboratory Animal Sciences, College of Basic medicine, Third Military Medical University, Chongqing 400038, China
| | - Zhidong Luan
- Department of Developmental Biology, Liaoning Medical University, Jinzhou, Liaoning 121001, China
| | - Miguel A Esteban
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Hongkui Deng
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing 100101, China
- Center for Life Sciences, Peking University, Beijing 100101, China
| | | | - Duanqing Pei
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
| | - Ning Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Gang Pei
- Tongji University, Shanghai 200092, China
| | - Lin Liu
- Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Yutao Du
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China
- BGI Ark Biotechnology Co., Ltd., Shenzhen, Guangdong 518083, China
| | - Lei Xiao
- College of Animal Sciences, Stem Cell and Developmental Biology Research Center, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Liangxue Lai
- Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China
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27
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Liu Z, Wan H, Wang E, Zhao X, Ding C, Zhou S, Li T, Shuai L, Feng C, Yu Y, Zhou Q, Beaujean N. Induced pluripotent stem-induced cells show better constitutive heterochromatin remodeling and developmental potential after nuclear transfer than their parental cells. Stem Cells Dev 2012; 21:3001-9. [PMID: 22657835 DOI: 10.1089/scd.2011.0646] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.
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Affiliation(s)
- Zichuan Liu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
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28
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Liu Z, Hai T, Dai X, Zhao X, Wang Y, Brochard V, Zhou S, Wan H, Zhang H, Wang L, Zhou Q, Beaujean N. Early patterning of cloned mouse embryos contributes to post-implantation development. Dev Biol 2012; 368:304-11. [PMID: 22659081 DOI: 10.1016/j.ydbio.2012.05.027] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2010] [Revised: 05/18/2012] [Accepted: 05/23/2012] [Indexed: 12/20/2022]
Abstract
Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.
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Affiliation(s)
- Zichuan Liu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
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29
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Deng Y, Liu Q, Luo C, Chen S, Li X, Wang C, Liu Z, Lei X, Zhang H, Sun H, Lu F, Jiang J, Shi D. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors. Stem Cells Dev 2012; 21:2485-94. [PMID: 22420535 DOI: 10.1089/scd.2012.0018] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.
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Affiliation(s)
- Yanfei Deng
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University , Nanning, People's Republic of China
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30
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Deshmukh RS, Østrup O, Strejcek F, Vejlsted M, Lucas-Hahn A, Petersen B, Li J, Callesen H, Niemann H, Hyttel P. Early aberrations in chromatin dynamics in embryos produced under in vitro conditions. Cell Reprogram 2012; 14:225-34. [PMID: 22468997 DOI: 10.1089/cell.2011.0069] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.
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Affiliation(s)
- Rahul S Deshmukh
- Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark
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31
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A Glimpse of Stem Cell Research in China. PROG BIOCHEM BIOPHYS 2011. [DOI: 10.3724/sp.j.1206.2011.00489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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32
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Ruan W, Han J, Li P, Cao S, An Y, Lim B, Li N. A novel strategy to derive iPS cells from porcine fibroblasts. SCIENCE CHINA. LIFE SCIENCES 2011; 54:553-9. [PMID: 21706416 DOI: 10.1007/s11427-011-4179-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/12/2011] [Accepted: 04/23/2011] [Indexed: 12/14/2022]
Abstract
Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.
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Affiliation(s)
- WeiMin Ruan
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
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