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1
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Tsukamoto M, Akutsu H. Comparative gastrointestinal organoid models across species: A Zoobiquity approach for precision medicine. Regen Ther 2025; 28:314-320. [PMID: 39885871 PMCID: PMC11779682 DOI: 10.1016/j.reth.2024.12.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2024] [Accepted: 12/20/2024] [Indexed: 02/01/2025] Open
Abstract
Gastrointestinal (GI) health underpins systemic well-being, yet the complexity of gut physiology poses significant challenges to understanding disease mechanisms and developing effective, personalized therapies. Traditional models often fail to capture the intricate interplay between epithelial, mesenchymal, immune, and neuronal cells that govern gut homeostasis and disease. Over the past five years, advances in organoid technology have created physiologically relevant, three-dimensional GI models that replicate native tissue architecture and function. These models have revolutionized the study of autoimmune disorders, homeostatic dysfunction, and pathogen infections, such as norovirus and Salmonella, which affect millions of humans and animals globally. In this review, we explore how organoids, derived from intestinal and pluripotent stem cells, are transforming our understanding of GI development, disease etiology, and therapeutic innovation. Through the "Zoobiquity" paradigm and "One Health" framework, we highlight the integration of companion animal organoids, which provide invaluable insights into shared disease mechanisms and preclinical therapeutic development. Despite their promise, challenges remain in achieving organoid maturation, expanding immune and neuronal integration, and bridging the gap between organoid responses and in vivo outcomes. By refining these cutting-edge platforms, we can advance human and veterinary medicine alike, fostering a holistic approach to health and disease.
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Affiliation(s)
- Masaya Tsukamoto
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Hidenori Akutsu
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
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2
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Menzorov AG. Pluripotent Stem Cells of Order Carnivora: Technical Perspective. Int J Mol Sci 2023; 24:ijms24043905. [PMID: 36835318 PMCID: PMC9963171 DOI: 10.3390/ijms24043905] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 02/08/2023] [Accepted: 02/12/2023] [Indexed: 02/17/2023] Open
Abstract
Human and mouse induced pluripotent stem cells (PSCs) are widely used for studying early embryonic development and for modeling of human diseases. Derivation and studying of PSCs from model organisms beyond commonly used mice and rats may provide new insights into the modeling and treating human diseases. The order Carnivora representatives possess unique features and are already used for modeling human-related traits. This review focuses on the technical aspects of derivation of the Carnivora species PSCs as well as their characterization. Current data on dog, feline, ferret, and American mink PSCs are summarized.
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Affiliation(s)
- Aleksei G. Menzorov
- Sector of Cell Collections, Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia;
- Natural Sciences Department, Novosibirsk State University, 630090 Novosibirsk, Russia
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3
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Olsson PO, Yeonwoo J, Park K, Yoo YM, Hwang WS. Live births from urine derived cells. PLoS One 2023; 18:e0278607. [PMID: 36696395 PMCID: PMC9876353 DOI: 10.1371/journal.pone.0278607] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Accepted: 11/21/2022] [Indexed: 01/26/2023] Open
Abstract
Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.
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Affiliation(s)
| | | | - Kyumi Park
- Department of Companion Animal & Animal Resources Science, Joongbu University, Geumsan-gun, Republic of Korea
| | - Yeong-Min Yoo
- Lab of Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, South Korea
| | - W. S. Hwang
- UAE Biotech Research Center, Abu Dhabi, UAE
- * E-mail:
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4
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Beklemisheva VR, Belokopytova PS, Fishman VS, Menzorov AG. Derivation of Ringed Seal ( Phoca hispida) Induced Multipotent Stem Cells. Cell Reprogram 2021; 23:326-335. [PMID: 34788122 DOI: 10.1089/cell.2021.0037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Induced pluripotent stem (iPS) cells have been produced just for a few species among order Carnivora: snow leopard, Bengal tiger, serval, jaguar, cat, dog, ferret, and American mink. We applied the iPS cell derivation protocol to the ringed seal (Phoca hispida) fibroblasts. The resulting cell line had the expression of pluripotency marker gene Rex1. Differentiation in embryoid body-like structures allowed us to register expression of AFP, endoderm marker, and Cdx2, trophectoderm marker, but not neuronal (ectoderm) markers. The cells readily differentiated into adipocytes and osteocytes, mesoderm cell types of origin. Transcriptome analysis allowed us to conclude that the cell line does not resemble human pluripotent cells, and, therefore, most probably is not pluripotent. Thus, we produced ringed seal multipotent stem cell line capable of differentiation into adipocytes and osteocytes.
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Affiliation(s)
- Violetta R Beklemisheva
- Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - Polina S Belokopytova
- Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - Veniamin S Fishman
- Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia
| | - Aleksei G Menzorov
- Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia
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5
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L- myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation. Cells 2021; 10:cells10081980. [PMID: 34440750 PMCID: PMC8391401 DOI: 10.3390/cells10081980] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 07/23/2021] [Accepted: 07/28/2021] [Indexed: 12/26/2022] Open
Abstract
Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.
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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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Kimura K, Tsukamoto M, Tanaka M, Kuwamura M, Ohtaka M, Nishimura K, Nakanishi M, Sugiura K, Hatoya S. Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells. Stem Cells Dev 2021; 30:79-90. [PMID: 33256572 DOI: 10.1089/scd.2020.0084] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.
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Affiliation(s)
- Kazuto Kimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Masaya Tsukamoto
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Miyuu Tanaka
- Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Mitsuru Kuwamura
- Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | | | - Ken Nishimura
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Mahito Nakanishi
- TOKIWA-Bio, Inc., Tsukuba, Japan
- National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
| | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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Questa M, Moshref M, Jimenez RJ, Lopez‐Cervantes V, Crawford CK, Settles ML, Ross PJ, Kol A. Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming. Stem Cells Transl Med 2020; 10:441-454. [PMID: 33210453 PMCID: PMC7900587 DOI: 10.1002/sctm.20-0278] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/15/2020] [Accepted: 10/09/2020] [Indexed: 12/17/2022] Open
Abstract
Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell-based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine-specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC-seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell-fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine-specific reprogramming approaches.
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Affiliation(s)
- Maria Questa
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
| | - Maryam Moshref
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
| | - Robert J. Jimenez
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
| | - Veronica Lopez‐Cervantes
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
| | - Charles K. Crawford
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
| | - Matthew L. Settles
- Bioinformatics Core FacilityUniversity of California DavisDavisCaliforniaUSA
| | - Pablo J. Ross
- Department of Animal ScienceUniversity of California DavisDavisCaliforniaUSA
| | - Amir Kol
- Department of Pathology, Microbiology and ImmunologySchool of Veterinary Medicine, University of California DavisDavisCaliforniaUSA
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9
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Menon DV, Bhaskar S, Sheshadri P, Joshi CG, Patel D, Kumar A. Positioning canine induced pluripotent stem cells (iPSCs) in the reprogramming landscape of naïve or primed state in comparison to mouse and human iPSCs. Life Sci 2020; 264:118701. [PMID: 33130086 DOI: 10.1016/j.lfs.2020.118701] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Revised: 10/25/2020] [Accepted: 10/28/2020] [Indexed: 12/11/2022]
Abstract
AIMS Deriving canine-induced pluripotent stem cells (ciPSCs) have paved the way for developing novel cell-based disease models and transplantation therapies in the dog. Though ciPSCs have been derived in the presence of Leukemia inhibitory factor (LIF) as well in the presence of basic fibroblast growth factor (bFGF), the positioning of ciPSCs in the naïve or the primed state of pluripotency remains elusive. This study aims to understand whether canine iPSCs belong to naïve or prime state in comparison to mouse (m) iPSCs and human (h) iPSCs. MAIN METHODS In the present study, we derived ciPSCs in presence of LIF and compared their state of pluripotency with that of miPSCs and hiPSCs by culturing them in the presence of LIF, bFGF, and LIF + bFGF. Gene expression level at transcript level was performed by RT-PCR and qRT-PCR and at the protein level was analysed by immunofluorescence. We also attempted to understand the pluripotency state using lipid body analysis by bodipy staining and blue fluorescence emission. KEY FINDINGS In contrast to miPSCs, the naïve pluripotent stem cells, ciPSCs showed the expression of FGF5 similar to that of primed pluripotent stem cell, hiPSCs. Compared to miPSCs, ciPSCs cultured in presence of LIF showed enhanced expression of primed pluripotent marker FGF5, similar to hiPSCs cultured in presence of bFGF. Upon culturing in hiPSC culture condition, ciPSCs showed enhanced expression of core pluripotency genes compared to miPSCs cultured in similar condition. However, ciPSCs expressed naïve pluripotent marker SSEA1 similar to miPSCs and lacked the expression of primed state marker SSEA4 unlike hiPSCs. Interestingly, for the first time, we demonstrate the ciPSC pluripotency using lipid body analysis wherein ciPSCs showed enhanced bodipy staining and blue fluorescence emission, reflecting the primed state of pluripotency. ciPSCs expressed higher levels of fatty acid synthase (FASN), the enzyme involved in the synthesis of palmitate, similar to that of hiPSCs and higher than that of miPSCs. As ciPSCs exhibit characteristic properties of both naïve and primed pluripotent state, it probably represents a unique intermediary state of pluripotency that is distinct from that of mice and human pluripotent stem cells. SIGNIFICANCE Elucidating the pluripotent state of ciPSCs assists in better understanding of the reprogramming events and development in different species. The study would provide a footprint of species-specific differences involved in reprogramming and the potential implication of iPSCs as a tool to analyse evolution.
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Affiliation(s)
- Dhanya V Menon
- Manipal Institute of Regenerative Medicine (MIRM), Manipal Academy of Higher Education, Bangalore, India; P.D.Patel Institute of Applied Sciences, Charusat University, Changa, Gujarat, India
| | - Smitha Bhaskar
- Manipal Institute of Regenerative Medicine (MIRM), Manipal Academy of Higher Education, Bangalore, India
| | - Preethi Sheshadri
- Manipal Institute of Regenerative Medicine (MIRM), Manipal Academy of Higher Education, Bangalore, India
| | - Chaitanya G Joshi
- Gujarat Biotechnology Research Centre, Department of Science and Technology, Gandhinagar, Gujarat, India
| | - Darshan Patel
- P.D.Patel Institute of Applied Sciences, Charusat University, Changa, Gujarat, India
| | - Anujith Kumar
- Manipal Institute of Regenerative Medicine (MIRM), Manipal Academy of Higher Education, Bangalore, India.
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Abstract
Early embryogenesis is characterized by the segregation of cell lineages that fulfill critical roles in the establishment of pregnancy and development of the fetus. The formation of the blastocyst marks the emergence of extraembryonic precursors, needed for implantation, and of pluripotent cells, which differentiate toward the major lineages of the adult organism. The coordinated emergence of these cell types shows that these processes are broadly conserved in mammals. However, developmental heterochrony and changes in gene regulatory networks highlight unique evolutionary adaptations that may explain the diversity in placentation and in the mechanisms controlling pluripotency in mammals. The incorporation of new technologies, including single-cell omics, imaging, and gene editing, is instrumental for comparative embryology. Broadening the knowledge of mammalian embryology will provide new insights into the mechanisms driving evolution and development. This knowledge can be readily translated into biomedical and biotechnological applications in humans and livestock, respectively.
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Affiliation(s)
- Ramiro Alberio
- School of Biosciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom;
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11
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Menon DV, Patel D, Joshi CG, Kumar A. The road less travelled: The efficacy of canine pluripotent stem cells. Exp Cell Res 2019; 377:94-102. [DOI: 10.1016/j.yexcr.2019.01.025] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Revised: 01/20/2019] [Accepted: 01/22/2019] [Indexed: 12/28/2022]
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12
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Barboni B, Russo V, Berardinelli P, Mauro A, Valbonetti L, Sanyal H, Canciello A, Greco L, Muttini A, Gatta V, Stuppia L, Mattioli M. Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance. Cell Transplant 2019; 27:93-116. [PMID: 29562773 PMCID: PMC6434480 DOI: 10.1177/0963689717724797] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The field of regenerative medicine is moving toward clinical practice in veterinary science. In this context, placenta-derived stem cells isolated from domestic animals have covered a dual role, acting both as therapies for patients and as a valuable cell source for translational models. The biological properties of placenta-derived cells, comparable among mammals, make them attractive candidates for therapeutic approaches. In particular, stemness features, low immunogenicity, immunomodulatory activity, multilineage plasticity, and their successful capacity for long-term engraftment in different host tissues after autotransplantation, allo-transplantation, or xenotransplantation have been demonstrated. Their beneficial regenerative effects in domestic animals have been proven using preclinical studies as well as clinical trials starting to define the mechanisms involved. This is, in particular, for amniotic-derived cells that have been thoroughly studied to date. The regenerative role arises from a mutual tissue-specific cell differentiation and from the paracrine secretion of bioactive molecules that ultimately drive crucial repair processes in host tissues (e.g., anti-inflammatory, antifibrotic, angiogenic, and neurogenic factors). The knowledge acquired so far on the mechanisms of placenta-derived stem cells in animal models represent the proof of concept of their successful use in some therapeutic treatments such as for musculoskeletal disorders. In the next future, legislation in veterinary regenerative medicine will be a key element in order to certify those placenta-derived cell-based protocols that have already demonstrated their safety and efficacy using rigorous approaches and to improve the degree of standardization of cell-based treatments among veterinary clinicians.
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Affiliation(s)
- B Barboni
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - V Russo
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - P Berardinelli
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - A Mauro
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - L Valbonetti
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - H Sanyal
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - A Canciello
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - L Greco
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - A Muttini
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - V Gatta
- 1 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - L Stuppia
- 2 Medical Genetics, University "G. d'Annunzio" of Chieti Pescara, Chieti, Italy
| | - M Mattioli
- 3 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale," Teramo, Italy
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13
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Lee J, Lee KS, Kim CL, Byeon JS, Gu NY, Cho IS, Cha SH. Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells. J Vet Sci 2018; 18:141-148. [PMID: 27456768 PMCID: PMC5489460 DOI: 10.4142/jvs.2017.18.2.141] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Revised: 04/19/2016] [Accepted: 06/08/2016] [Indexed: 01/08/2023] Open
Abstract
Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.
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Affiliation(s)
- Jienny Lee
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - Keum Sil Lee
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - Chan-Lan Kim
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - Jeong Su Byeon
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - Na-Yeon Gu
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - In-Soo Cho
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
| | - Sang-Ho Cha
- Viral Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
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14
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Ramos-Ibeas P, Nichols J, Alberio R. States and Origins of Mammalian Embryonic Pluripotency In Vivo and in a Dish. Curr Top Dev Biol 2017; 128:151-179. [PMID: 29477162 DOI: 10.1016/bs.ctdb.2017.11.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Mouse embryonic stem cells (ESC), derived from preimplantation embryos in 1981, defined mammalian pluripotency for many decades. However, after the derivation of human ESC in 1998, comparative studies showed that different types of pluripotency exist in early embryos and that these can be captured in vitro under various culture conditions. Over the past decade much has been learned about the key signaling pathways, growth factor requirements, and transcription factor profiles of pluripotent cells in embryos, allowing improvement of derivation and culture conditions for novel pluripotent stem cell types. More recently, studies using single-cell transcriptomics of embryos from different species provided an unprecedented level of resolution of cellular interactions and cell fate decisions that are informing new ways to understand the emergence of pluripotency in different organisms. These new approaches enhance knowledge of species differences during early embryogenesis and will be instrumental for improving methodologies for generating intra- and interspecies chimeric animals using pluripotent stem cells. Here, we discuss the recent developments in our understanding of early embryogenesis in different mammalian species.
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Affiliation(s)
| | - Jennifer Nichols
- Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom; University of Cambridge, Cambridge, United Kingdom.
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Nottingham, United Kingdom.
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15
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Paterson YZ, Kafarnik C, Guest DJ. Characterization of companion animal pluripotent stem cells. Cytometry A 2017; 93:137-148. [PMID: 28678404 DOI: 10.1002/cyto.a.23163] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 05/19/2017] [Accepted: 06/10/2017] [Indexed: 02/06/2023]
Abstract
Pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. These properties underpin their potential to be used in regenerative medicine. Originally derived from early embryos, pluripotent stem cells can now be derived by reprogramming an adult cell back to a pluripotent state. Companion animals such as horses, dogs, and cats suffer from many injuries and diseases for which regenerative medicine may offer new treatments. As many of the injuries and diseases are similar to conditions in humans the use of companion animals for the experimental and clinical testing of stem cell and regenerative medicine products would provide relevant animal models for the translation of therapies to the human field. In order to fully utilize companion animal pluripotent stem cells robust, standardized methods of characterization must be developed to ensure that safe and effective treatments can be delivered. In this review we discuss the methods that are available for characterizing pluripotent stem cells and the techniques that have been applied in cells from companion animals. We describe characteristics which have been described consistently across reports as well as highlighting discrepant results. Significant steps have been made to define the in vitro culture requirements and drive lineage specific differentiation of pluripotent stem cells in companion animal species. However, additional basic research to compare pluripotent stem cell types and define characteristics of pluripotency in companion animal species is still required. © 2017 International Society for Advancement of Cytometry.
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Affiliation(s)
- Y Z Paterson
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, UK.,Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
| | - C Kafarnik
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, UK.,Institute of Ophthalmology, University College London, London, UK
| | - D J Guest
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, UK
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16
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Nishimura T, Unezaki N, Kanegi R, Wijesekera DPH, Hatoya S, Sugiura K, Kawate N, Tamada H, Imai H, Inaba T. Generation of Canine Induced Extraembryonic Endoderm-Like Cell Line That Forms Both Extraembryonic and Embryonic Endoderm Derivatives. Stem Cells Dev 2017; 26:1111-1120. [PMID: 28474540 DOI: 10.1089/scd.2017.0026] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.
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Affiliation(s)
- Toshiya Nishimura
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Naoya Unezaki
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Ryoji Kanegi
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | | | - Shingo Hatoya
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Kikuya Sugiura
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Noritoshi Kawate
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Hiromichi Tamada
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
| | - Hiroshi Imai
- 2 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, Japan
| | - Toshio Inaba
- 1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Osaka, Japan
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17
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Nishimura T, Hatoya S, Kanegi R, Wijesekera DPH, Sanno K, Tanaka E, Sugiura K, Hiromitsu Tamada NK, Imai H, Inaba T. Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions. Mol Reprod Dev 2017; 84:329-339. [PMID: 28240438 DOI: 10.1002/mrd.22789] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 02/09/2017] [Indexed: 12/29/2022]
Abstract
Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.
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Affiliation(s)
- Toshiya Nishimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Ryoji Kanegi
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | | | - Kousuke Sanno
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Erina Tanaka
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | | | - Hiroshi Imai
- Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Toshio Inaba
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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18
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Pluripotency of embryo-derived stem cells from rodents, lagomorphs, and primates: Slippery slope, terrace and cliff. Stem Cell Res 2017; 19:104-112. [DOI: 10.1016/j.scr.2017.01.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Revised: 01/01/2017] [Accepted: 01/13/2017] [Indexed: 12/14/2022] Open
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19
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Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts. Theriogenology 2017; 92:75-82. [PMID: 28237347 DOI: 10.1016/j.theriogenology.2017.01.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2016] [Revised: 11/18/2016] [Accepted: 01/06/2017] [Indexed: 12/13/2022]
Abstract
Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.
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20
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Luo J, Cibelli JB. Conserved Role of bFGF and a Divergent Role of LIF for Pluripotency Maintenance and Survival in Canine Pluripotent Stem Cells. Stem Cells Dev 2016; 25:1670-1680. [DOI: 10.1089/scd.2016.0164] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Affiliation(s)
- Jiesi Luo
- Department of Animal Science, Michigan State University, East Lansing, Michigan
- Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut
| | - Jose B. Cibelli
- Department of Animal Science, Michigan State University, East Lansing, Michigan
- Department of Physiology, Michigan State University, East Lansing, Michigan
- LARCEL, Laboratorio Andaluz de Reprogramación Celular, BIONAND, Andalucía, Spain
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21
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Tobias IC, Brooks CR, Teichroeb JH, Villagómez DA, Hess DA, Séguin CA, Betts DH. Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency. Stem Cells Dev 2016; 25:1208-22. [DOI: 10.1089/scd.2016.0103] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Affiliation(s)
- Ian C. Tobias
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
| | - Courtney R. Brooks
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
| | - Jonathan H. Teichroeb
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
| | - Daniel A. Villagómez
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
- Departamento de Producción Animal, Universidad de Guadalajara, Zapopan, Jalisco, Mexico
| | - David A. Hess
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
- Children's Health Research Institute, the University of Western Ontario, London, Ontario, Canada
- Molecular Medicine Research Group, Krembil Centre for Stem Cell Biology, Robarts Research Institute, the University of Western Ontario, London, Ontario Canada
| | - Cheryle A. Séguin
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
- Children's Health Research Institute, the University of Western Ontario, London, Ontario, Canada
| | - Dean H. Betts
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario, London, Ontario, Canada
- Children's Health Research Institute, the University of Western Ontario, London, Ontario, Canada
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22
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Brevini T, Pennarossa G, Acocella F, Brizzola S, Zenobi A, Gandolfi F. Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells. Vet J 2016; 211:52-6. [DOI: 10.1016/j.tvjl.2016.02.014] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2015] [Revised: 01/29/2016] [Accepted: 02/27/2016] [Indexed: 12/15/2022]
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23
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Menzorov AG, Matveeva NM, Markakis MN, Fishman VS, Christensen K, Khabarova AA, Pristyazhnyuk IE, Kizilova EA, Cirera S, Anistoroaei R, Serov OL. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes. BMC Genomics 2015; 16 Suppl 13:S6. [PMID: 26694224 PMCID: PMC4686781 DOI: 10.1186/1471-2164-16-s13-s6] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Background Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. Results We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. Conclusions We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.
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24
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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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25
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Betts DH, Tobias IC. Canine Pluripotent Stem Cells: Are They Ready for Clinical Applications? Front Vet Sci 2015; 2:41. [PMID: 26664969 PMCID: PMC4672225 DOI: 10.3389/fvets.2015.00041] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2015] [Accepted: 09/21/2015] [Indexed: 12/20/2022] Open
Abstract
The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner.
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Affiliation(s)
- Dean H Betts
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario , London, ON , Canada ; Children's Health Research Institute, Lawson Health Research Institute , London, ON , Canada
| | - Ian C Tobias
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario , London, ON , Canada
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26
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Baird A, Barsby T, Guest DJ. Derivation of Canine Induced Pluripotent Stem Cells. Reprod Domest Anim 2015; 50:669-76. [PMID: 26074059 DOI: 10.1111/rda.12562] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Accepted: 05/23/2015] [Indexed: 12/31/2022]
Abstract
Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose-derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re-programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c-myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re-activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.
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Affiliation(s)
- Aeg Baird
- Animal Health Trust, Kentford, Newmarket, Suffolk, UK
| | - T Barsby
- Animal Health Trust, Kentford, Newmarket, Suffolk, UK
| | - D J Guest
- Animal Health Trust, Kentford, Newmarket, Suffolk, UK
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27
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Gonçalves NN, Ambrósio CE, Piedrahita JA. Stem Cells and Regenerative Medicine in Domestic and Companion Animals: A Multispecies Perspective. Reprod Domest Anim 2014; 49 Suppl 4:2-10. [DOI: 10.1111/rda.12392] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2014] [Accepted: 07/14/2014] [Indexed: 12/18/2022]
Affiliation(s)
- NN Gonçalves
- Department of Veterinary Medicine; Faculty of Animal Science and Food Engineering; FZEA/USP; Pirassununga Sao Paulo Brazil
- Department of Surgery; Faculty of Veterinary Medicine and Animal Science; FMVZ/USP; Sao Paulo Brazil
| | - CE Ambrósio
- Department of Veterinary Medicine; Faculty of Animal Science and Food Engineering; FZEA/USP; Pirassununga Sao Paulo Brazil
- Department of Surgery; Faculty of Veterinary Medicine and Animal Science; FMVZ/USP; Sao Paulo Brazil
| | - JA Piedrahita
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine; North Carolina State University; Raleigh NC USA
- Center for Comparative Medicine and Translational Research; North Carolina State University; Raleigh NC USA
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28
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Whitworth DJ, Banks TA. Stem cell therapies for treating osteoarthritis: prescient or premature? Vet J 2014; 202:416-24. [PMID: 25457267 DOI: 10.1016/j.tvjl.2014.09.024] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2013] [Revised: 09/22/2014] [Accepted: 09/25/2014] [Indexed: 01/23/2023]
Abstract
There has been unprecedented interest in recent years in the use of stem cells as therapy for an array of diseases in companion animals. Stem cells have already been deployed therapeutically in a number of clinical settings, in particular the use of mesenchymal stem cells to treat osteoarthritis in horses and dogs. However, an assessment of the scientific literature highlights a marked disparity between the purported benefits of stem cell therapies and their proven abilities as defined by rigorously controlled scientific studies. Although preliminary data generated from clinical trials in human patients are encouraging, therapies currently available to treat animals are supported by very limited clinical evidence, and the commercialisation of these treatments may be premature. This review introduces the three main types of stem cells relevant to veterinary applications, namely, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells, and draws together research findings from in vitro and in vivo studies to give an overview of current stem cell therapies for the treatment of osteoarthritis in animals. Recent advances in tissue engineering, which is proposed as the future direction of stem cell-based therapy for osteoarthritis, are also discussed.
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Affiliation(s)
- Deanne J Whitworth
- School of Veterinary Science, University of Queensland, Gatton, Queensland 4343, Australia.
| | - Tania A Banks
- School of Veterinary Science, University of Queensland, Gatton, Queensland 4343, Australia
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29
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Koh S, Piedrahita JA. From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals. Theriogenology 2014; 81:103-11. [PMID: 24274415 DOI: 10.1016/j.theriogenology.2013.09.009] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2013] [Revised: 09/05/2013] [Accepted: 09/05/2013] [Indexed: 01/10/2023]
Abstract
Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as "embryonic stem-like" cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs.
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Affiliation(s)
- Sehwon Koh
- Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, USA; Genomics Program, North Carolina State University, Raleigh, North Carolina, USA
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30
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Lee KS, Cha SH, Kang HW, Song JY, Lee KW, Ko KB, Lee HT. Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived mesenchymal stem cells. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2014; 26:588-95. [PMID: 25049827 PMCID: PMC4093376 DOI: 10.5713/ajas.2012.12488] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Revised: 12/20/2012] [Accepted: 11/13/2012] [Indexed: 12/27/2022]
Abstract
Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.
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Affiliation(s)
- K S Lee
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - S-H Cha
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - H W Kang
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - J-Y Song
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - K W Lee
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - K B Ko
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
| | - H T Lee
- Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Korea
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Duggal G, Heindryckx B, Deroo T, De Sutter P. Use of pluripotent stem cells for reproductive medicine: are we there yet? Vet Q 2014; 34:42-51. [DOI: 10.1080/01652176.2014.891061] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
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Lee KS, Kang HW, Lee HT, Kim HJ, Kim CL, Song JY, Lee KW, Cha SH. Sequential sub-passage decreases the differentiation potential of canine adipose-derived mesenchymal stem cells. Res Vet Sci 2013; 96:267-75. [PMID: 24447790 DOI: 10.1016/j.rvsc.2013.12.011] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2013] [Revised: 11/20/2013] [Accepted: 12/22/2013] [Indexed: 01/01/2023]
Abstract
Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.
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Affiliation(s)
- Keum Sil Lee
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea; Department of Animal Biotechnology, KonKuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea
| | - Hye Won Kang
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea
| | - Hoon Taek Lee
- Department of Animal Biotechnology, KonKuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea
| | - Hye-Jin Kim
- Haemaru Referral Animal Hospital, Bundang-gu, Seongnam-si, Gyeonggi-do 463-824, Republic of Korea
| | - Chan-Lan Kim
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea
| | - Jae-Young Song
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea
| | - Kyung Woo Lee
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
| | - Sang-Ho Cha
- Animal and Plant Quarantine Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
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Hall V, Hinrichs K, Lazzari G, Betts DH, Hyttel P. Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals. Vet J 2013; 197:128-42. [PMID: 23810186 DOI: 10.1016/j.tvjl.2013.05.026] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Revised: 05/03/2013] [Accepted: 05/04/2013] [Indexed: 01/01/2023]
Abstract
Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.
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Affiliation(s)
- V Hall
- Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Denmark
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34
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Volk SW, Theoret C. Translating stem cell therapies: the role of companion animals in regenerative medicine. Wound Repair Regen 2013; 21:382-94. [PMID: 23627495 DOI: 10.1111/wrr.12044] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2012] [Accepted: 01/30/2013] [Indexed: 12/24/2022]
Abstract
Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell-based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the "One Health Initiative," the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.
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Affiliation(s)
- Susan W Volk
- Department of Clinical Studies and Animal Biology, School of Veterinary Medicine, The University of Pennsylvania, Philadelphia 19104-4539, USA.
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35
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Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells. Cytotechnology 2013; 66:239-50. [PMID: 23553019 DOI: 10.1007/s10616-013-9561-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2012] [Accepted: 03/26/2013] [Indexed: 10/27/2022] Open
Abstract
The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.
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36
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Nishimura T, Hatoya S, Kanegi R, Sugiura K, Wijewardana V, Kuwamura M, Tanaka M, Yamate J, Izawa T, Takahashi M, Kawate N, Tamada H, Imai H, Inaba T. Generation of functional platelets from canine induced pluripotent stem cells. Stem Cells Dev 2013; 22:2026-35. [PMID: 23409943 DOI: 10.1089/scd.2012.0701] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.
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Affiliation(s)
- Toshiya Nishimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Japan
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Tobias IC, Brooks CR, Teichroeb JH, Betts DH. Derivation and culture of canine embryonic stem cells. Methods Mol Biol 2013; 1074:69-83. [PMID: 23975806 DOI: 10.1007/978-1-62703-628-3_6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
The derivation of canine embryonic stem cells (cESCs) represents a significant achievement and opens the door to further stem cell research and therapies in the dog. Canines share a common environment with humans and exhibit a host of genetic diseases, many of which have human parallels. Thus, the canine model presents unique advantages over other currently used organisms to help develop stem cell therapies in humans. To reveal the therapeutic potential of cESCs further basic research on the molecular mechanisms controlling their pluripotency and self-renewal characteristics is needed. Herein, we present the methods for derivation and culture of cESCs. Following collection of the canine blastocyst, two derivation methods are presented; immunodissection and whole blastocyst explant. These two methods lead to cESCs differing in morphology and subculture techniques. Additional protocols for subculture of established lines, feeder-free culture, and cryopreservation protocols are also described.
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Affiliation(s)
- Ian C Tobias
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada
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38
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Lim JH, Koh S, Olby NJ, Piedrahita J, Mariani CL. Isolation and characterization of neural progenitor cells from adult canine brains. Am J Vet Res 2012; 73:1963-8. [DOI: 10.2460/ajvr.73.12.1963] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Koh S, Thomas R, Tsai S, Bischoff S, Lim JH, Breen M, Olby NJ, Piedrahita JA. Growth requirements and chromosomal instability of induced pluripotent stem cells generated from adult canine fibroblasts. Stem Cells Dev 2012; 22:951-63. [PMID: 23016947 DOI: 10.1089/scd.2012.0393] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.
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Affiliation(s)
- Sehwon Koh
- Genomics Program, North Carolina State University, Raleigh, NC 27607, USA
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40
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Breton A, Sharma R, Diaz AC, Parham AG, Graham A, Neil C, Whitelaw CB, Milne E, Donadeu FX. Derivation and characterization of induced pluripotent stem cells from equine fibroblasts. Stem Cells Dev 2012; 22:611-21. [PMID: 22897112 DOI: 10.1089/scd.2012.0052] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine.
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Affiliation(s)
- Amandine Breton
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
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Lee KS, Nah JJ, Lee BC, Lee HT, Lee HS, So BJ, Cha SH. Maintenance and characterization of multipotent mesenchymal stem cells isolated from canine umbilical cord matrix by collagenase digestion. Res Vet Sci 2012; 94:144-51. [PMID: 22975229 DOI: 10.1016/j.rvsc.2012.07.033] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Revised: 06/20/2012] [Accepted: 07/28/2012] [Indexed: 10/27/2022]
Abstract
Multipotent mesenchymal stem cells have been considered as a novel clinical approach for cell therapy and regenerative medicine. In this study, mesenchymal stem cells (MSCs) were successfully isolated from canine umbilical cord matrix (cUCM; also referred to as Wharton's Jelly) by collagenase digestion and further characterized for multipotent properties associated with MSCs. Our cUCM-derived MSCs (cUCM-MSCs) were plastic adherent, spindle-shaped and fibroblast-like cells, maintaining expression of pluripotency markers such as Oct3/4, Nanog, Sox-2 and SSEA-4 as well as normal chromosomal number during a long-term proliferative culture. The cells expressed MSCs-specific surface markers, including CD44, CD90, CD105, and CD184, but did not CD29, CD33, CD34, and CD45. More importantly, cUCM-MSCs could differentiate into mesodermal (adipocyte, osteocyte and chondrocyte) and ectodermal (neuronal cell) cell lineages. These results imply that collagenase digestion would be a highly effective way to isolate multipotent MSCs in abundant amounts.
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Affiliation(s)
- K S Lee
- Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 6-dong, Anyang-si, Gyeonggi-do 430-757, Republic of Korea
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Takemitsu H, Zhao D, Yamamoto I, Harada Y, Michishita M, Arai T. Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells. BMC Vet Res 2012; 8:150. [PMID: 22937862 PMCID: PMC3442961 DOI: 10.1186/1746-6148-8-150] [Citation(s) in RCA: 99] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2012] [Accepted: 08/15/2012] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis. RESULTS Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs. CONCLUSION Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.
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Affiliation(s)
- Hiroshi Takemitsu
- Department of Veterinary Science, School of Veterinary medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonancho, Musashino, Tokyo 180-8602, Japan
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Xue F, Ma Y, Chen YE, Zhang J, Lin TA, Chen CH, Lin WW, Roach M, Ju JC, Yang L, Du F, Xu J. Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells. Cell Reprogram 2012; 14:364-76. [PMID: 22775411 DOI: 10.1089/cell.2012.0001] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Abstract
The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.
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Affiliation(s)
- Fei Xue
- Renova Life Inc., University of Maryland, TAP program, College Park, MD 20740, USA
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Nishida H, Nakayama M, Tanaka H, Kitamura M, Hatoya S, Sugiura K, Harada Y, Suzuki Y, Ide C, Inaba T. Safety of autologous bone marrow stromal cell transplantation in dogs with acute spinal cord injury. Vet Surg 2012; 41:437-42. [PMID: 22548465 DOI: 10.1111/j.1532-950x.2011.00959.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
OBJECTIVE To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). STUDY DESIGN An open-label single-arm trial. ANIMALS Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. METHODS Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dog's femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. RESULTS Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. CONCLUSIONS Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.
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Goldhawk DE, Rohani R, Sengupta A, Gelman N, Prato FS. Using the magnetosome to model effective gene-based contrast for magnetic resonance imaging. WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY 2012; 4:378-88. [DOI: 10.1002/wnan.1165] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
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Whitworth DJ, Ovchinnikov DA, Wolvetang EJ. Generation and characterization of LIF-dependent canine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev 2012; 21:2288-97. [PMID: 22221227 DOI: 10.1089/scd.2011.0608] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Dogs provide a more clinically relevant model of human disease than rodents, particularly with respect to hereditary diseases. Thus, the availability of canine stem cells will greatly facilitate the use of the dog in the development of stem cell-based gene therapies and regenerative medicine. In this study we describe the production of canine induced pluripotent stem cells (ciPSCs) from adult dermal fibroblasts. These cells have a morphology resembling previously described canine embryonic stem cells, a normal karyotype, and express pluripotency markers including alkaline phosphatase, Nanog, Oct4, Telomerase, SSEA1, SSEA4, TRA1-60, TRA1-81, and Rex1. Furthermore, the inactive X chromosome is reactivated indicating a ground-state pluripotency. In culture they readily form embryoid bodies, which in turn give rise to cell types from all 3 embryonic germ layers, as indicated by expression of the definitive endoderm markers Cxcr4 and α-fetoprotein, mesoderm markers Collagen IIA and Gata2, and ectoderm markers βIII-tubulin, Enolase, and Nestin. Of particular significance is the observation that these ciPSCs are dependent only on leukemia inhibitory factor (LIF), making them similar to mouse and canine embryonic stem cells, but strikingly unlike the ciPSCs recently described in two other studies, which were dependent on both basic fibroblast growth factor and LIF in order to maintain their pluripotency. Thus, our ciPSCs closely resemble mouse ESCs derived from the inner cell mass of preimplantation embryos, while the previously described ciPSCs appear to be more representative of cells from the epiblast of mouse postimplantation embryos.
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Affiliation(s)
- Deanne J Whitworth
- School of Veterinary Science, University of Queensland, Gatton, Queensland, Australia.
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47
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Gattegno-Ho D, Argyle SA, Argyle DJ. Stem cells and veterinary medicine: Tools to understand diseases and enable tissue regeneration and drug discovery. Vet J 2012; 191:19-27. [DOI: 10.1016/j.tvjl.2011.08.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2011] [Revised: 08/08/2011] [Accepted: 08/09/2011] [Indexed: 01/21/2023]
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Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays? Arch Toxicol 2011; 85:1361-72. [DOI: 10.1007/s00204-011-0767-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2011] [Accepted: 10/06/2011] [Indexed: 01/31/2023]
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Back to basics: stem cells and veterinary medicine. Vet J 2011; 191:139-40. [PMID: 21723756 DOI: 10.1016/j.tvjl.2011.05.023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2011] [Accepted: 05/27/2011] [Indexed: 11/21/2022]
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Lee AS, Xu D, Plews JR, Nguyen PK, Nag D, Lyons JK, Han L, Hu S, Lan F, Liu J, Huang M, Narsinh KH, Long CT, de Almeida PE, Levi B, Kooreman N, Bangs C, Pacharinsak C, Ikeno F, Yeung AC, Gambhir SS, Robbins RC, Longaker MT, Wu JC. Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells. J Biol Chem 2011; 286:32697-704. [PMID: 21719696 DOI: 10.1074/jbc.m111.235739] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.
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Affiliation(s)
- Andrew S Lee
- Department of Radiology, Stanford University School of Medicine, Stanford, California 94305-5454, USA
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