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Neira JA, Conrad JV, Rusteika M, Chu LF. The progress of induced pluripotent stem cells derived from pigs: a mini review of recent advances. Front Cell Dev Biol 2024; 12:1371240. [PMID: 38979033 PMCID: PMC11228285 DOI: 10.3389/fcell.2024.1371240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 04/10/2024] [Indexed: 07/10/2024] Open
Abstract
Pigs (Sus scrofa) are widely acknowledged as an important large mammalian animal model due to their similarity to human physiology, genetics, and immunology. Leveraging the full potential of this model presents significant opportunities for major advancements in the fields of comparative biology, disease modeling, and regenerative medicine. Thus, the derivation of pluripotent stem cells from this species can offer new tools for disease modeling and serve as a stepping stone to test future autologous or allogeneic cell-based therapies. Over the past few decades, great progress has been made in establishing porcine pluripotent stem cells (pPSCs), including embryonic stem cells (pESCs) derived from pre- and peri-implantation embryos, and porcine induced pluripotent stem cells (piPSCs) using a variety of cellular reprogramming strategies. However, the stabilization of pPSCs was not as straightforward as directly applying the culture conditions developed and optimized for murine or primate PSCs. Therefore, it has historically been challenging to establish stable pPSC lines that could pass stringent pluripotency tests. Here, we review recent advances in the establishment of stable porcine PSCs. We focus on the evolving derivation methods that eventually led to the establishment of pESCs and transgene-free piPSCs, as well as current challenges and opportunities in this rapidly advancing field.
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Affiliation(s)
- Jaime A Neira
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
| | - J Vanessa Conrad
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
| | - Margaret Rusteika
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
- Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada
| | - Li-Fang Chu
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
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2
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Conrad JV, Meyer S, Ramesh PS, Neira JA, Rusteika M, Mamott D, Duffin B, Bautista M, Zhang J, Hiles E, Higgins EM, Steill J, Freeman J, Ni Z, Liu S, Ungrin M, Rancourt D, Clegg DO, Stewart R, Thomson JA, Chu LF. Efficient derivation of transgene-free porcine induced pluripotent stem cells enables in vitro modeling of species-specific developmental timing. Stem Cell Reports 2023; 18:2328-2343. [PMID: 37949072 PMCID: PMC10724057 DOI: 10.1016/j.stemcr.2023.10.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 10/10/2023] [Accepted: 10/10/2023] [Indexed: 11/12/2023] Open
Abstract
Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ∼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.
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Affiliation(s)
- J Vanessa Conrad
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada
| | - Susanne Meyer
- Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Pranav S Ramesh
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada
| | - Jaime A Neira
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada; Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Margaret Rusteika
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Daniel Mamott
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - Bret Duffin
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - Monica Bautista
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada
| | - Jue Zhang
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - Emily Hiles
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada
| | - Eve M Higgins
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada
| | - John Steill
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - Jack Freeman
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - Zijian Ni
- Department of Statistics, University of Wisconsin, Madison, WI 53706, USA
| | - Shiying Liu
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Mark Ungrin
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Derrick Rancourt
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada; Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Dennis O Clegg
- Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; Department of Molecular, Cellular, & Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Ron Stewart
- Morgridge Institute for Research, Madison, WI 53715, USA
| | - James A Thomson
- Morgridge Institute for Research, Madison, WI 53715, USA; Department of Molecular, Cellular, & Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Li-Fang Chu
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada; Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
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Species-Specific Enhancer Activity of OCT4 in Porcine Pluripotency: The Porcine OCT4 Reporter System Could Monitor Pluripotency in Porcine Embryo Development and Embryonic Stem Cells. Stem Cells Int 2022; 2022:6337532. [PMID: 35846983 PMCID: PMC9277468 DOI: 10.1155/2022/6337532] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Accepted: 05/06/2022] [Indexed: 01/31/2023] Open
Abstract
The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.
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4
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Chakritbudsabong W, Chaiwattanarungruengpaisan S, Sariya L, Pamonsupornvichit S, Ferreira JN, Sukho P, Gronsang D, Tharasanit T, Dinnyes A, Rungarunlert S. Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells. Front Cell Dev Biol 2021; 9:709286. [PMID: 34354993 PMCID: PMC8329718 DOI: 10.3389/fcell.2021.709286] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Accepted: 06/18/2021] [Indexed: 12/20/2022] Open
Abstract
Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.
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Affiliation(s)
- Warunya Chakritbudsabong
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.,Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.,Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Somjit Chaiwattanarungruengpaisan
- The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MOZWE), Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Ladawan Sariya
- The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MOZWE), Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Sirikron Pamonsupornvichit
- The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MOZWE), Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Joao N Ferreira
- Exocrine Gland Biology and Regeneration Research Group, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
| | - Panithi Sukho
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.,Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Dulyatad Gronsang
- Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
| | - Theerawat Tharasanit
- Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Andras Dinnyes
- BioTalentum Ltd., Gödöllő, Hungary.,Department of Physiology and Animal Health, Institute of Physiology and Animal Health, Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary.,College of Life Sciences, Sichuan University, Chengdu, China
| | - Sasitorn Rungarunlert
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.,Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
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5
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Setthawong P, Phakdeedindan P, Techakumphu M, Tharasanit T. Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells. Reprod Domest Anim 2021; 56:1104-1116. [PMID: 34013645 DOI: 10.1111/rda.13954] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Accepted: 05/17/2021] [Indexed: 12/29/2022]
Abstract
Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.
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Affiliation(s)
- Piyathip Setthawong
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Praopilas Phakdeedindan
- Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Mongkol Techakumphu
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Theerawat Tharasanit
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.,CU-Animal Fertility Research Unit, Chulalongkorn University, Bangkok, Thailand
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6
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Su Y, Zhu J, Salman S, Tang Y. Induced pluripotent stem cells from farm animals. J Anim Sci 2021; 98:5937369. [PMID: 33098420 DOI: 10.1093/jas/skaa343] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023] Open
Abstract
The development of the induced pluripotent stem cells (iPSCs) technology has revolutionized the world on the establishment of pluripotent stem cells (PSCs) across a great variety of animal species. Generation of iPSCs from domesticated animals would provide unrestricted cell resources for the study of embryonic development and cell differentiation of these species, for screening and establishing desired traits for sustainable agricultural production, and as veterinary and preclinical therapeutic tools for animal and human diseases. Induced PSCs from domesticated animals thus harbor enormous scientific, economical, and societal values. Although much progress has been made toward the generation of PSCs from these species, major obstacles remain precluding the exclamation of the establishment of bona fide iPSCs. The most prominent of them remain the inability of these cells to silence exogenous reprogramming factors, the obvious reliance on exogenous factors for their self-renewal, and the restricted development potential in vivo. In this review, we summarize the history and current progress in domestic farm animal iPSC generation, with a focus on swine, ruminants (cattle, ovine, and caprine), horses, and avian species (quails and chickens). We also discuss the problems associated with the farm animal iPSCs and potential future directions toward the complete reprogramming of somatic cells from farm animals.
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Affiliation(s)
- Yue Su
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Jiaqi Zhu
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Saleh Salman
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
| | - Young Tang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT
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7
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Scarfone RA, Pena SM, Russell KA, Betts DH, Koch TG. The use of induced pluripotent stem cells in domestic animals: a narrative review. BMC Vet Res 2020; 16:477. [PMID: 33292200 PMCID: PMC7722595 DOI: 10.1186/s12917-020-02696-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 11/24/2020] [Indexed: 02/07/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) are undifferentiated stem cells characterized by the ability to differentiate into any cell type in the body. iPSCs are a relatively new and rapidly developing technology in many fields of biology, including developmental anatomy and physiology, pathology, and toxicology. These cells have great potential in research as they are self-renewing and pluripotent with minimal ethical concerns. Protocols for their production have been developed for many domestic animal species, which have since been used to further our knowledge in the progression and treatment of diseases. This research is valuable both for veterinary medicine as well as for the prospect of translation to human medicine. Safety, cost, and feasibility are potential barriers for this technology that must be considered before widespread clinical adoption. This review will analyze the literature pertaining to iPSCs derived from various domestic species with a focus on iPSC production and characterization, applications for tissue and disease research, and applications for disease treatment.
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Affiliation(s)
- Rachel A Scarfone
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Samantha M Pena
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Keith A Russell
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
| | - Dean H Betts
- Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada
| | - Thomas G Koch
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada.
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8
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Cong X, Zhang SM, Ellis MW, Luo J. Large Animal Models for the Clinical Application of Human Induced Pluripotent Stem Cells. Stem Cells Dev 2019; 28:1288-1298. [PMID: 31359827 DOI: 10.1089/scd.2019.0136] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Induced pluripotent stem cell (iPSC) technology offers a practically infinite and ethically acceptable source to obtain a variety of somatic cells. Coupled with the biotechnologies of cell therapy or tissue engineering, iPSC technology will enormously contribute to human regenerative medicine. Before clinical application, such human iPSC (hiPSC)-based therapies should be assessed using large animal models that more closely match biological or biomechanical properties of human patients. Therefore, it is critical to generate large animal iPSCs, obtain their iPSC-derived somatic cells, and preclinically evaluate their therapeutic efficacy and safety in large animals. During the past decade, the establishment of iPSC lines of a series of large animal species has been documented, and the acquisition and preclinical evaluation of iPSC-derived somatic cells has also been reported. Despite this progress, significant obstacles, such as obtaining or preserving the bona fide pluripotency of large animal iPSCs, have been encountered. Simultaneously, studies of large animal iPSCs have been overlooked in comparison with those of mouse and hiPSCs, and this field deserves more attention and support due to its important preclinical relevance. Herein, this review will focus on the large animal models of pigs, dogs, horses, and sheep/goats, and summarize current progress, challenges, and potential future directions of research on large animal iPSCs.
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Affiliation(s)
- Xiaoqiang Cong
- Section of Cardiovascular Medicine, Department of Internal Medicine, Yale Cardiovascular Research Center, Yale School of Medicine, New Haven, Connecticut.,Department of Cardiology, Bethune First Hospital of Jilin University, Changchun, China
| | - Shang-Min Zhang
- Department of Pathology, Yale School of Medicine, New Haven, Connecticut
| | - Matthew W Ellis
- Section of Cardiovascular Medicine, Department of Internal Medicine, Yale Cardiovascular Research Center, Yale School of Medicine, New Haven, Connecticut.,Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut
| | - Jiesi Luo
- Section of Cardiovascular Medicine, Department of Internal Medicine, Yale Cardiovascular Research Center, Yale School of Medicine, New Haven, Connecticut.,Yale Stem Cell Center, New Haven, Connecticut
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9
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Pessôa LVDF, Bressan FF, Freude KK. Induced pluripotent stem cells throughout the animal kingdom: Availability and applications. World J Stem Cells 2019; 11:491-505. [PMID: 31523369 PMCID: PMC6716087 DOI: 10.4252/wjsc.v11.i8.491] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2019] [Revised: 06/18/2019] [Accepted: 06/20/2019] [Indexed: 02/06/2023] Open
Abstract
Up until the mid 2000s, the capacity to generate every cell of an organism was exclusive to embryonic stem cells. In 2006, researchers Takahashi and Yamanaka developed an alternative method of generating embryonic-like stem cells from adult cells, which they coined induced pluripotent stem cells (iPSCs). Such iPSCs possess most of the advantages of embryonic stem cells without the ethical stigma associated with derivation of the latter. The possibility of generating “custom-made” pluripotent cells, ideal for patient-specific disease models, alongside their possible applications in regenerative medicine and reproduction, has drawn a lot of attention to the field with numbers of iPSC studies published growing exponentially. IPSCs have now been generated for a wide variety of species, including but not limited to, mouse, human, primate, wild felines, bovines, equines, birds and rodents, some of which still lack well-established embryonic stem cell lines. The paucity of robust characterization of some of these iPSC lines as well as the residual expression of transgenes involved in the reprogramming process still hampers the use of such cells in species preservation or medical research, underscoring the requirement for further investigations. Here, we provide an extensive overview of iPSC generated from a broad range of animal species including their potential applications and limitations.
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Affiliation(s)
- Laís Vicari de Figueiredo Pessôa
- Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg 1870, Denmark
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga 13635-000, São Paulo, Brazil
| | - Kristine Karla Freude
- Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg 1870, Denmark
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10
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Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa. Stem Cells Int 2019; 2019:2130973. [PMID: 30992705 PMCID: PMC6434273 DOI: 10.1155/2019/2130973] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2018] [Revised: 12/31/2018] [Accepted: 01/14/2019] [Indexed: 01/30/2023] Open
Abstract
OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.
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11
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Setthawong P, Phakdeedindan P, Tiptanavattana N, Rungarunlert S, Techakumphu M, Tharasanit T. Generation of porcine induced-pluripotent stem cells from Sertoli cells. Theriogenology 2018; 127:32-40. [PMID: 30639694 DOI: 10.1016/j.theriogenology.2018.12.033] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Revised: 12/18/2018] [Accepted: 12/20/2018] [Indexed: 01/04/2023]
Abstract
Induced pluripotent stem cells (iPSCs) are generated by reprogramming of somatic cells using four transcription factors: OCT4, SOX2, KLF-4, and c-MYC (OSKM). However, reprogramming efficiency of iPSCs is currently poor. In this study, we used the Sertoli line as a novel cell source for somatic cell reprogramming. Neonatal testes were collected from 1-week-old piglets. The testes were digested by a two-step enzymatic method to isolate Sertoli cells. The latter were transfected with retroviral vectors expressing OSKM. The Sertoli iPSC-like colonies were subjected to morphological analysis, alkaline phosphatase staining, RT-PCR, G-banding karyotyping, in vitro differentiation, and in vivo differentiation. Primary Sertoli cells had polygon-shaped morphology and manifested phagocytic activity as determined by a fluorescent bead assay. Sertoli cells also expressed the anti-Müllerian hormone protein in the cytoplasm. According to RT-PCR results, these cells expressed Sertoli cell markers (FSHR, KRT18, and GATA6) and endogenous transcription factors genes (KLF4 and c-MYC). A total of 240 colonies (0.3% efficiency) were detected by day 7 after viral transduction of 72500 cells. The Sertoli iPSC-like colonies contained small cells with a high nucleus-to-cytoplasm ratio. These colonies tested positive for alkaline phosphatase staining, expressed endogenous pluripotency genes, and had a normal karyotype. All these cell lines could form in vitro three-dimensional aggregates that represented three germ layers of embryonic-like cells. A total of two cell lines used for in vivo differentiation produced high-efficiency teratoma. In conclusion, Sertoli cells can efficiently serve as a novel cell source for iPSC reprogramming.
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Affiliation(s)
- Piyathip Setthawong
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
| | - Praopilas Phakdeedindan
- Biochemistry Unit, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
| | - Narong Tiptanavattana
- Faculty of Veterinary Science, Prince of Songkla University, Songkhla 90110, Thailand
| | - Sasitorn Rungarunlert
- Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73710, Thailand
| | - Mongkol Techakumphu
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
| | - Theerawat Tharasanit
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
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12
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Sun WS, Chun JL, Kim DH, Ahn JS, Kim MK, Hwang IS, Kwon DJ, Hwang S, Lee JW. Molecular cloning and characterization of porcine ribosomal protein L21. J Vet Sci 2018; 18:531-540. [PMID: 28057907 PMCID: PMC5746447 DOI: 10.4142/jvs.2017.18.4.531] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2016] [Revised: 10/12/2016] [Accepted: 11/23/2016] [Indexed: 11/20/2022] Open
Abstract
Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein has an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. In addition, we examined its expression pattern and function by using overexpression or knockdown approaches. As a result, we obtained a 604 bp segment that contains a 483 bp open reading frame encoding 160 amino acids. The pig RPL21 gene is located in the “+” strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in ovary and lung, at lower levels in kidney, small intestine, and skin, and at the lowest levels in heart and liver. Furthermore, RPL21 expression is closely connected with cell proliferation and cell cycle arrest. The results are intended to provide useful information for the further study of pig RPL21.
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Affiliation(s)
- Wu-Sheng Sun
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea.,Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea
| | - Ju-Lan Chun
- Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea
| | - Dong-Hwan Kim
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Jin-Seop Ahn
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Min-Kyu Kim
- Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea
| | - In-Sul Hwang
- Animal Biotechnology Division, National Institute of Animal Science, Wanju 55365, Korea
| | - Dae-Jin Kwon
- Animal Biotechnology Division, National Institute of Animal Science, Wanju 55365, Korea
| | - Seongsoo Hwang
- Animal Biotechnology Division, National Institute of Animal Science, Wanju 55365, Korea
| | - Jeong-Woong Lee
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
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13
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Secher JO, Ceylan A, Mazzoni G, Mashayekhi K, Li T, Muenthaisong S, Nielsen TT, Li D, Li S, Petkov S, Cirera S, Luo Y, Thombs L, Kadarmideen HN, Dinnyes A, Bolund L, Roelen BAJ, Schmidt M, Callesen H, Hyttel P, Freude KK. Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells. Mol Reprod Dev 2017; 84:229-245. [PMID: 28044390 PMCID: PMC6221014 DOI: 10.1002/mrd.22771] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2016] [Accepted: 12/16/2016] [Indexed: 12/14/2022]
Abstract
Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF‐derived piPSCs were more successful than their FGF‐derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole‐transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency‐related gene network of the LIF‐ versus FGF‐derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC‐like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC‐like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC‐like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line‐transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229–245, 2017. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Jan O Secher
- Veterinary Reproduction and Obstetrics, Faculty of Health and Medical Sciences, Department of Large Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Ahmet Ceylan
- Faculty of Veterinary Medicine Ankara University, Department of Histology and Embryology, Diskapi, Ankara, Turkey
| | - Gianluca Mazzoni
- Animal Breeding, Quantitative Genetics and Systems Biology Group, Faculty of Health and Medical Sciences, Department of Large Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Kaveh Mashayekhi
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark.,BioTalentum Ltd., Gödöllő, Hungary.,Faculty of Veterinary Medicine, Departments of Equine Sciences and Farm Animal Health, Utrecht University, Utrecht, Netherlands
| | - Tong Li
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark.,BioTalentum Ltd., Gödöllő, Hungary.,Faculty of Veterinary Medicine, Departments of Equine Sciences and Farm Animal Health, Utrecht University, Utrecht, Netherlands
| | - Suchitra Muenthaisong
- BioTalentum Ltd., Gödöllő, Hungary.,Faculty of Veterinary Medicine, Department of Farm Animal Health, Utrecht University, Utrecht, Netherlands
| | - Troels T Nielsen
- Danish Dementia Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - Dong Li
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Shengting Li
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark
| | - Stoyan Petkov
- Institute for Farm Animal Genetics (FLI), Neustadt, Germany
| | - Susanna Cirera
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Yonglun Luo
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark
| | - Lori Thombs
- Department of Statistics, University of Missouri, Columbia, Missouri
| | - Haja N Kadarmideen
- Animal Breeding, Quantitative Genetics and Systems Biology Group, Faculty of Health and Medical Sciences, Department of Large Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Andras Dinnyes
- BioTalentum Ltd., Gödöllő, Hungary.,Faculty of Veterinary Medicine, Departments of Equine Sciences and Farm Animal Health, Utrecht University, Utrecht, Netherlands.,Molecular Animal Biotechnology Laboratory, Szent István University, Gödöllő, Hungary
| | - Lars Bolund
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark
| | - Bernard A J Roelen
- Faculty of Veterinary Medicine, Department of Farm Animal Health, Utrecht University, Utrecht, Netherlands
| | - Mette Schmidt
- Veterinary Reproduction and Obstetrics, Faculty of Health and Medical Sciences, Department of Large Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Henrik Callesen
- Department of Animal Science, Aarhus University, Tjele, Denmark
| | - Poul Hyttel
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Kristine K Freude
- Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
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14
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Kwon DJ, Hwang IS, Kwak TU, Yang H, Park MR, Ock SA, Oh KB, Woo JS, Im GS, Hwang S. Effects of Cell Cycle Regulators on the Cell Cycle Synchronization of Porcine induced Pluripotent Stem Cells. Dev Reprod 2017; 21:47-54. [PMID: 28484743 PMCID: PMC5409209 DOI: 10.12717/dr.2017.21.1.047] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2017] [Revised: 02/15/2017] [Accepted: 02/20/2017] [Indexed: 11/24/2022]
Abstract
Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced
pluripotent stem cells (piPSCs) is very low. The present study was performed to
investigate the effect of cell cycle inhibitors on the cell cycle
synchronization of piPSCs. piPSCs were generated using combination of six human
transcriptional factors under stem cell culture condition. To examine the
efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel
coated plate with stem cell media and they were treated with staurosporine (STA,
20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO,
200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were
in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells
at G1 in DAI group was significantly higher than that in control, while STA,
ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of
viability and apoptosis were affected by STA and ROSC treatment, but there were
no significantly differences between control and DAI groups. Real-Time qPCR and
FACs results revealed that DAI treatment did not affect the expression of
pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and
apoptosis, but Oct4 expression was significantly decreased. Our results suggest
that DAI could be used for synchronizing piPSCs at G1 stage and has any
deleterious effect on survival and pluripotency sustaining of piPSCs.
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Affiliation(s)
- Dae-Jin Kwon
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea.,International Agricultural Development and Cooperation Center, Chonbuk National University, Jeonju 54896, Korea
| | - In-Sul Hwang
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Tae-Uk Kwak
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Hyeon Yang
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Mi-Ryung Park
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Sun-A Ock
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Keon Bong Oh
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Jae-Seok Woo
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Gi-Sun Im
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
| | - Seongsoo Hwang
- Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju 55365, Korea
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15
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DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells. PLoS One 2017; 12:e0173047. [PMID: 28253351 PMCID: PMC5333863 DOI: 10.1371/journal.pone.0173047] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Accepted: 02/14/2017] [Indexed: 01/05/2023] Open
Abstract
Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFβ pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.
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16
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Choi KH, Park JK, Son D, Hwang JY, Lee DK, Ka H, Park J, Lee CK. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig. PLoS One 2016; 11:e0158046. [PMID: 27336671 PMCID: PMC4918974 DOI: 10.1371/journal.pone.0158046] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2016] [Accepted: 06/09/2016] [Indexed: 12/22/2022] Open
Abstract
Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig.
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Affiliation(s)
- Kwang-Hwan Choi
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea
| | - Jin-Kyu Park
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri, United States of America
| | - Dongchan Son
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea
| | - Jae Yeon Hwang
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea
| | - Dong-Kyung Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea
| | - Hakhyun Ka
- Department of Biological Resources and Technology, Yonsei University, Wonju, Korea
| | | | - Chang-Kyu Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea
- Institute of Green Bio Science and Technology, Seoul National University, Pyeong Chang, Kangwon do, Korea
- * E-mail:
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17
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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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18
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Schook LB, Collares TV, Darfour-Oduro KA, De AK, Rund LA, Schachtschneider KM, Seixas FK. Unraveling the swine genome: implications for human health. Annu Rev Anim Biosci 2016; 3:219-44. [PMID: 25689318 DOI: 10.1146/annurev-animal-022114-110815] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The pig was first used in biomedical research in ancient Greece and over the past few decades has quickly grown into an important biomedical research tool. Pigs have genetic and physiological traits similar to humans, which make them one of the most useful and versatile animal models. Owing to these similarities, data generated from porcine models are more likely to lead to viable human treatments than those from murine work. In addition, the similarity in size and physiology to humans allows pigs to be used for many experimental approaches not feasible in mice. Research areas that employ pigs range from neonatal development to translational models for cancer therapy. Increasing numbers of porcine models are being developed since the release of the swine genome sequence, and the development of additional porcine genomic and epigenetic resources will further their use in biomedical research.
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Affiliation(s)
- Lawrence B Schook
- Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801; , , , ,
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19
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Abstract
This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably stem from an inability to maintain the stability of the endogenous gene networks necessary to maintain pluripotency. Based on work in humans and rodents, achievement of full pluripotency will likely require fine adjustments in the growth factors and signaling inhibitors provided to the cells. Finally, we discuss the future utility of these cells for biomedical and agricultural purposes.
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Affiliation(s)
- Toshihiko Ezashi
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - Ye Yuan
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
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20
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No JG, Choi MK, Kwon DJ, Yoo JG, Yang BC, Park JK, Kim DH. Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming. J Reprod Dev 2015; 61:90-8. [PMID: 25736622 PMCID: PMC4410095 DOI: 10.1262/jrd.2014-078] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.
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Affiliation(s)
- Jin-Gu No
- Animal Biotechnology Division; Department of Biological Science, Sungkyunkwan University, Suwon 440-746, Republic of Korea
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21
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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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Oct4 overexpression facilitates proliferation of porcine fibroblasts and development of cloned embryos. ZYGOTE 2014; 23:704-11. [PMID: 25181424 DOI: 10.1017/s0967199414000355] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.
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