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Yamaguchi N, Horio E, Sonoda J, Yamagishi M, Miyakawa S, Murakami F, Hasegawa H, Katahira Y, Mizoguchi I, Fujii Y, Chikazu D, Yoshimoto T. Immortalization of Mesenchymal Stem Cells for Application in Regenerative Medicine and Their Potential Risks of Tumorigenesis. Int J Mol Sci 2024; 25:13562. [PMID: 39769322 PMCID: PMC11676347 DOI: 10.3390/ijms252413562] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 12/13/2024] [Accepted: 12/16/2024] [Indexed: 01/11/2025] Open
Abstract
Regenerative medicine utilizes stem cells to repair damaged tissues by replacing them with their differentiated cells and activating the body's inherent regenerative abilities. Mesenchymal stem cells (MSCs) are adult stem cells that possess tissue repair and regenerative capabilities and immunomodulatory properties with a much lower risk of tumorigenicity, making them a focus of numerous clinical trials worldwide. MSCs primarily exert their therapeutic effects through paracrine effects via secreted factors, such as cytokines and exosomes. This has led to increasing interest in cell-free therapy, where only the conditioned medium (also called secretome) from MSC cultures is used for regenerative applications. However, MSCs face certain limitations, including cellular senescence, scarcity, donor heterogeneity, complexity, short survival post-implantation, and regulatory and ethics hurdles. To address these challenges, various types of immortalized MSCs (ImMSCs) capable of indefinite expansion have been developed. These cells offer significant promise and essential tools as a reliable source for both cell-based and cell-free therapies with the aim of translating them into practical medicine. However, the process of immortalization, often involving the transduction of immortalizing genes, poses potential risks of genetic instability and resultant malignant transformation. Cell-free therapy is particularly attractive, as it circumvents the risks of tumorigenicity and ethical concerns associated with live cell therapies. Rigorous safety tests, such as monitoring chromosomal abnormalities, are critical to ensure safety. Technologies like inducible or suicide genes may allow for the controlled proliferation of MSCs and induce apoptosis after their therapeutic task is completed. This review highlights recent advancements in the immortalization of MSCs and the associated risks of tumorigenesis.
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Affiliation(s)
- Natsuki Yamaguchi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Eri Horio
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Jukito Sonoda
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Miu Yamagishi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Satomi Miyakawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Fumihiro Murakami
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Hideaki Hasegawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yasuhiro Katahira
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Izuru Mizoguchi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yasuyuki Fujii
- Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
| | - Daichi Chikazu
- Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
| | - Takayuki Yoshimoto
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
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Lenz LS, Wink MR. The other side of the coin: mesenchymal stromal cell immortalization beyond evasion of senescence. Hum Cell 2023; 36:1593-1603. [PMID: 37341871 DOI: 10.1007/s13577-023-00925-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2023] [Accepted: 05/23/2023] [Indexed: 06/22/2023]
Abstract
Mesenchymal stromal cells (MSC) are promising options to cellular therapy to several clinical disorders, mainly because of its ability to immunomodulate and differentiate into different cell types. Even though MSC can be isolated from different sources, a major challenge to understanding the biological effects is that the primary cells undergo replicative senescence after a limited number of cell divisions in culture, requiring time-consuming and technically challenging approaches to get a sufficient cell number for clinical applications. Therefore, a new isolation, characterization, and expansion is necessary every time, which increases the variability and is time-consuming. Immortalization is a strategy that can overcome these challenges. Therefore, here, we review the different methodologies available to cellular immortalization, and discuss the literature regarding MSC immortalization and the broader biological consequences that extend beyond the mere increase in proliferation potential.
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Affiliation(s)
- Luana Suéling Lenz
- Laboratório de Biologia Celular, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, 90050-170, Brazil
| | - Márcia Rosângela Wink
- Laboratório de Biologia Celular, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, 90050-170, Brazil.
- Departamento de Ciências Básicas da Saúde (DCBS), Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, 90050-170, Brazil.
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Voloshin N, Tyurin-Kuzmin P, Karagyaur M, Akopyan Z, Kulebyakin K. Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives. Int J Mol Sci 2023; 24:12716. [PMID: 37628897 PMCID: PMC10454025 DOI: 10.3390/ijms241612716] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Revised: 07/23/2023] [Accepted: 08/09/2023] [Indexed: 08/27/2023] Open
Abstract
In modern science, immortalized cells are not only a convenient tool in fundamental research, but they are also increasingly used in practical medicine. This happens due to their advantages compared to the primary cells, such as the possibility to produce larger amounts of cells and to use them for longer periods of time, the convenience of genetic modification, the absence of donor-to-donor variability when comparing the results of different experiments, etc. On the other hand, immortalization comes with drawbacks: possibilities of malignant transformation and/or major phenotype change due to genetic modification itself or upon long-term cultivation appear. At first glance, such issues are huge hurdles in the way of immortalized cells translation into medicine. However, there are certain ways to overcome such barriers that we describe in this review. We determined four major areas of usage of immortalized cells for practical medicinal purposes, and each has its own means to negate the drawbacks associated with immortalization. Moreover, here we describe specific fields of application of immortalized cells in which these problems are of much lesser concern, for example, in some cases where the possibility of malignant growth is not there at all. In general, we can conclude that immortalized cells have their niches in certain areas of practical medicine where they can successfully compete with other therapeutic approaches, and more preclinical and clinical trials with them should be expected.
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Affiliation(s)
- Nikita Voloshin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Pyotr Tyurin-Kuzmin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Maxim Karagyaur
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Zhanna Akopyan
- Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia;
| | - Konstantin Kulebyakin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
- Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia;
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Zhang L, Wan Z, Yuan Z, Yang J, Zhang Y, Cai Q, Huang J, Zhao Y. Construction of multifunctional cell aggregates in angiogenesis and osteogenesis through incorporating hVE-cad-Fc-modified PLGA/β-TCP microparticles for enhancing bone regeneration. J Mater Chem B 2022; 10:3344-3356. [PMID: 35380570 DOI: 10.1039/d2tb00359g] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Multicellular aggregates have been widely utilized for regenerative medicine; however, the heterogeneous structure and undesired bioactivity of cell-only aggregates hinder their clinical translation. In this study, we fabricated an innovative kind of microparticle-integrated cellular aggregate with multifunctional activities in angiogenesis and osteogenesis, by combining stem cells from human exfoliated deciduous teeth (SHEDs) and bioactive composite microparticles. The poly(lactide-co-glycolide) (PLGA)-based bioactive microparticles (PTV microparticles) were ∼15 μm in diameter, with dispersed β-tricalcium phosphate (β-TCP) nanoparticles and surface-modified vascular endothelialcadherin fusion protein (hVE-cad-Fc). After co-culturing with microparticles in U-bottomed culture plates, SHEDs could firmly attach to the microparticles with a homogeneous distribution. The PTV microparticle-integrated SHED aggregates (PTV/SHED aggregates) showed significant positive CD31 and ALP expression, as well as the significantly upregulated osteogenesis makers (Runx2, ALP, and OCN) and angiogenesis makers (Ang-1 and CD31), compared with PLGA, PLGA/β-TCP (PT) and PLGA/hVE-cad-Fc (PV) microparticle-integrated SHED aggregates. Finally, in mice, 3 mm calvarial defects filled with the PTV microparticle-integrated SHED aggregates achieved abundant vascularized neo-bone regeneration within 4 weeks. Overall, we believe that these multifunctional PTV/SHED aggregates could be used as modules for bottom-up regenerative medicine, and provide a promising method for vascularized bone regeneration.
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Affiliation(s)
- Linxue Zhang
- Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology & Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health & NMPA Key Laboratory for Dental Materials, Beijing 100081, PR China.
| | - Zhuo Wan
- State Key Laboratory of Organic-Inorganic Composites & Beijing Laboratory of Biomedical Materials & Beijing University of Chemical Technology, Beijing 100029, PR China. .,Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, PR China.
| | - Zuoying Yuan
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, PR China.
| | - Jun Yang
- The Key Laboratory of Bioactive Materials, Ministry of Education & College of Life Science, Nankai University, Tianjin 300071, PR China
| | - Yunfan Zhang
- Department of Orthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology & Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health & NMPA Key Laboratory for Dental Materials, Beijing 100081, PR China
| | - Qing Cai
- State Key Laboratory of Organic-Inorganic Composites & Beijing Laboratory of Biomedical Materials & Beijing University of Chemical Technology, Beijing 100029, PR China.
| | - Jianyong Huang
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, PR China.
| | - Yuming Zhao
- Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology & Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health & NMPA Key Laboratory for Dental Materials, Beijing 100081, PR China.
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Beckenkamp LR, da Fontoura DMS, Korb VG, de Campos RP, Onzi GR, Iser IC, Bertoni APS, Sévigny J, Lenz G, Wink MR. Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity. Stem Cell Rev Rep 2021; 16:776-791. [PMID: 32556945 DOI: 10.1007/s12015-020-09986-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.
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Affiliation(s)
- L R Beckenkamp
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil
| | - D M S da Fontoura
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil
| | - V G Korb
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil
| | - R P de Campos
- Department of Biophysics and Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
| | - G R Onzi
- Department of Biophysics and Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
| | - I C Iser
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil
| | - A P S Bertoni
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil
| | - J Sévigny
- Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Québec city, QC, G1V 0A6, Canada.,Centre de recherche du CHU de Québec, Université Laval, Québec city, QC, G1V 4G2, Canada
| | - G Lenz
- Department of Biophysics and Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
| | - Márcia Rosângela Wink
- Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil.
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6
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Okuchi Y, Reeves J, Ng SS, Doro DH, Junyent S, Liu KJ, El Haj AJ, Habib SJ. Wnt-modified materials mediate asymmetric stem cell division to direct human osteogenic tissue formation for bone repair. NATURE MATERIALS 2021; 20:108-118. [PMID: 32958876 DOI: 10.1038/s41563-020-0786-5] [Citation(s) in RCA: 44] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/18/2019] [Accepted: 07/27/2020] [Indexed: 06/11/2023]
Abstract
The maintenance of human skeletal stem cells (hSSCs) and their progeny in bone defects is a major challenge. Here, we report on a transplantable bandage containing a three-dimensional Wnt-induced osteogenic tissue model (WIOTM). This bandage facilitates the long-term viability of hSSCs (8 weeks) and their progeny, and enables bone repair in an in vivo mouse model of critical-sized calvarial defects. The newly forming bone is structurally comparable to mature cortical bone and consists of human and murine cells. Furthermore, we show that the mechanism of WIOTM formation is governed by Wnt-mediated asymmetric cell division of hSSCs. Covalently immobilizing Wnts onto synthetic materials can polarize single dividing hSSCs, orient the spindle and simultaneously generate a Wnt-proximal hSSC and a differentiation-prone Wnt-distal cell. Our results provide insight into the regulation of human osteogenesis and represent a promising approach to deliver human osteogenic constructs that can survive in vivo and contribute to bone repair.
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Affiliation(s)
- Yoshihisa Okuchi
- Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK
| | - Joshua Reeves
- Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK
| | - Soon Seng Ng
- Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK
| | - Daniel H Doro
- Centre for Craniofacial and Regenerative Biology, King's College London, London, UK
| | - Sergi Junyent
- Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK
| | - Karen J Liu
- Centre for Craniofacial and Regenerative Biology, King's College London, London, UK
| | - Alicia J El Haj
- Healthcare Technology Institute, Institute of Translational Medicine, University of Birmingham, Birmingham, UK
| | - Shukry J Habib
- Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK.
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7
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Tyurin-Kuzmin PA, Chechekhin VI, Ivanova AM, Dyikanov DT, Sysoeva VY, Kalinina NI, Tkachuk VA. Noradrenaline Sensitivity Is Severely Impaired in Immortalized Adipose-Derived Mesenchymal Stem Cell Line. Int J Mol Sci 2018; 19:ijms19123712. [PMID: 30469522 PMCID: PMC6321284 DOI: 10.3390/ijms19123712] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Revised: 11/15/2018] [Accepted: 11/20/2018] [Indexed: 01/20/2023] Open
Abstract
Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called “heterologous sensitization”, and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca2+-mobilization in single cells, we found that these cells did not demonstrate the sensitization seen in primary adMSCs. Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology.
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Affiliation(s)
- Pyotr A Tyurin-Kuzmin
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Vadim I Chechekhin
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Anastasiya M Ivanova
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Daniyar T Dyikanov
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Veronika Y Sysoeva
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Natalia I Kalinina
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
| | - Vsevolod A Tkachuk
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia.
- Laboratory of Molecular Endocrinology, Russian Cardiology Research Center, 121552 Moscow, Russia.
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Peng X, Zhang Y, Wang Y, He Q, Yu Q. IGF‐1 and BMP‐7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone‐marrow mesenchymal stem cells. J Cell Biochem 2018; 120:5570-5582. [PMID: 30417423 DOI: 10.1002/jcb.27841] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2018] [Accepted: 09/14/2018] [Indexed: 12/15/2022]
Affiliation(s)
- Xian‐Bo Peng
- Department of Joint Surgery & Sports Medicine Qianfoshan Hospital of Shandong Province Jinan China
| | - Yuan Zhang
- Department of Geriatric Neurology Qianfoshan Hospital of Shandong Province Jinan China
| | - Yue‐Qiu Wang
- Department of Joint Branch Jining No. 2 People’s Hospital, Shandong Province Jining China
| | - Qi He
- Department of Blood Transfusion Shandong Provincial Hospital Jinan China
| | - Qian Yu
- Department of Joint Surgery & Sports Medicine Qianfoshan Hospital of Shandong Province Jinan China
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Murphy MP, Quarto N, Longaker MT, Wan DC. * Calvarial Defects: Cell-Based Reconstructive Strategies in the Murine Model. Tissue Eng Part C Methods 2017; 23:971-981. [PMID: 28825366 DOI: 10.1089/ten.tec.2017.0230] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Calvarial defects pose a continued clinical dilemma for reconstruction. Advancements within the fields of stem cell biology and tissue engineering have enabled researchers to develop reconstructive strategies using animal models. We review the utility of various animal models and focus on the mouse, which has aided investigators in understanding cranial development and calvarial bone healing. The murine model has also been used to study regenerative approaches to critical-sized calvarial defects, and we discuss the application of stem cells such as bone marrow-derived mesenchymal stromal cells, adipose-derived stromal cells, muscle-derived stem cells, and pluripotent stem cells to address deficient bone in this animal. Finally, we highlight strategies to manipulate stem cells using various growth factors and inhibitors and ultimately how these factors may prove crucial in future advancements within calvarial reconstruction using native skeletal stem cells.
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Affiliation(s)
- Matthew P Murphy
- 1 Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University , Stanford, California.,2 Lorry I. Lokey Stem Cell Research Building, Stanford Stem Cell Biology and Regenerative Medicine Institute, Stanford University , Stanford, California
| | - Natalina Quarto
- 1 Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University , Stanford, California
| | - Michael T Longaker
- 1 Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University , Stanford, California.,2 Lorry I. Lokey Stem Cell Research Building, Stanford Stem Cell Biology and Regenerative Medicine Institute, Stanford University , Stanford, California
| | - Derrick C Wan
- 1 Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University , Stanford, California
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10
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Trachana V, Petrakis S, Fotiadis Z, Siska EK, Balis V, Gonos ES, Kaloyianni M, Koliakos G. Human mesenchymal stem cells with enhanced telomerase activity acquire resistance against oxidative stress-induced genomic damage. Cytotherapy 2017; 19:808-820. [PMID: 28454681 DOI: 10.1016/j.jcyt.2017.03.078] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2017] [Revised: 03/25/2017] [Accepted: 03/25/2017] [Indexed: 12/15/2022]
Abstract
BACKGROUND Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. METHODS Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). RESULTS We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. DISCUSSION On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell's basic antioxidant machinery.
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Affiliation(s)
- Varvara Trachana
- Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41500 Larisa, Greece.
| | - Spyros Petrakis
- Biohellenika Biotechnology Company, 57001 Thessaloniki, Greece
| | - Zisis Fotiadis
- Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Evangelia K Siska
- Biohellenika Biotechnology Company, 57001 Thessaloniki, Greece; Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Vasileios Balis
- Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Efstathios S Gonos
- National Hellenic Research Foundation, 48 Vas. Konstantinou Str, 11635 Athens, Greece
| | - Martha Kaloyianni
- Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - George Koliakos
- Biohellenika Biotechnology Company, 57001 Thessaloniki, Greece; Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
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Zhu X, Liu Z, Deng W, Zhang Z, Liu Y, Wei L, Zhang Y, Zhou L, Wang Y. Derivation and characterization of sheep bone marrow-derived mesenchymal stem cells induced with telomerase reverse transcriptase. Saudi J Biol Sci 2017; 24:519-525. [PMID: 28386176 PMCID: PMC5372373 DOI: 10.1016/j.sjbs.2017.01.022] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2016] [Revised: 12/23/2016] [Accepted: 01/06/2017] [Indexed: 02/06/2023] Open
Abstract
Bone marrow mesenchymal stem cells (BMSCs) are a type of adult stem cells with a wide range of potential applications. However, BMSCs have a limited life cycle under normal culturing conditions, which has hindered further study and application. Many studies have confirmed that cells modified by telomerase reverse transcriptase (TERT) can maintain the ability to proliferate in vitro over a long period of time. In this study, we constructed a gene expression vector to transfer TERT into sheep BMSCs, and evaluated whether the TERT cell strain was successfully transferred. The abilities of cell proliferation and differentiation were evaluated using the methods including growth curve determination, inheritance stability analysis, multi-directional induction and so on, and the results showed that the cell strain can be cultured to 40 generations, with a normal karyotype rate maintained at 88.24%, and that the cell strain can be transferred and differentiated into neurocytes and lipocytes, proving that it retains the multi-directional transdifferentiation ability.
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Affiliation(s)
- Xuemin Zhu
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Zongzheng Liu
- Animal Husbandry and Veterinary Research Institute of Qingdao, Qingdao 266000, China
| | - Wen Deng
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Ziqiang Zhang
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Yumei Liu
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Lan Wei
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Yuling Zhang
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Liutao Zhou
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
| | - Yuzhu Wang
- College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
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12
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Cai YD, Zhang Q, Zhang YH, Chen L, Huang T. Identification of Genes Associated with Breast Cancer Metastasis to Bone on a Protein–Protein Interaction Network with a Shortest Path Algorithm. J Proteome Res 2017; 16:1027-1038. [DOI: 10.1021/acs.jproteome.6b00950] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Yu-Dong Cai
- School
of Life Sciences, Shanghai University, Shanghai 200444 People’s Republic of China
| | - Qing Zhang
- School
of Life Sciences, Shanghai University, Shanghai 200444 People’s Republic of China
| | - Yu-Hang Zhang
- Institute
of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
| | - Lei Chen
- College
of Information Engineering, Shanghai Maritime University, Shanghai 201306, People’s Republic of China
| | - Tao Huang
- Institute
of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
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13
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Ravindran S, George A. Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration. Front Physiol 2015; 6:118. [PMID: 25954205 PMCID: PMC4404808 DOI: 10.3389/fphys.2015.00118] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Accepted: 03/28/2015] [Indexed: 12/20/2022] Open
Abstract
Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world's population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues.
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Affiliation(s)
- Sriram Ravindran
- Department of Oral Biology, University of Illinois at Chicago Chicago, IL, USA
| | - Anne George
- Department of Oral Biology, University of Illinois at Chicago Chicago, IL, USA
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14
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Li C, Wei G, Gu Q, Wang Q, Tao S, Xu L. Proliferation and differentiation of rat osteoporosis mesenchymal stem cells (MSCs) after telomerase reverse transcriptase (TERT) transfection. Med Sci Monit 2015; 21:845-54. [PMID: 25796354 PMCID: PMC4381855 DOI: 10.12659/msm.893144] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Background The aim of this study was to determine whether MSC are excellent materials for MSCs transplantation in the treatment of osteoporosis. Material/Methods We studied normal, osteoporosis, and TERT-transfected MSC from normal and osteoporosis rats to compare the proliferation and osteogenic differentiation using RT-PCR and Western blot by constructing an ovariectomized rat model of osteoporosis (OVX). The primary MSC from model rats were extracted and cultured to evaluate the proliferation and differentiation characteristics. Results MSCs of osteoporosis rats obviously decreased in proliferation ability and osteogenic differentiation compared to that of normal rats. In contrast, in TERT-transfected MSC, the proliferation and differentiation ability, and especially the ability of osteogenic differentiation, were significantly higher than in osteoporosis MSC. Conclusions TERT-transfected MSCs can help osteoporosis patients in whom MSC proliferation and osteogenic differentiation ability are weak, with an increase in both bone mass and bone density, becoming an effective material for autologous transplantation of MSCs in further treatment of osteoporosis. However, studies are still needed to prove the in vivo effect, biological safety, and molecular mechanism of TERT-osteoporosis treatment. Additionally, because the results are from an animal model, more research is needed in generalizing rat model findings to human osteoporosis patients.
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Affiliation(s)
- Chao Li
- Department of Orthopaedics, 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China (mainland)
| | - Guojun Wei
- Department of Orthopaedics, 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China (mainland)
| | - Qun Gu
- Department of Orthopaedics, 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China (mainland)
| | - Qiang Wang
- Department of Orthopaedics, Affiliated Hospital of School of Medicine of Ningbo University, Ningbo, Zhejiang, China (mainland)
| | - Shuqin Tao
- Department of Orthopaedics, 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China (mainland)
| | - Liang Xu
- Department of Orthopaedics, 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China (mainland)
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15
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Nagao-Kitamoto H, Setoguchi T, Kitamoto S, Nakamura S, Tsuru A, Nagata M, Nagano S, Ishidou Y, Yokouchi M, Kitajima S, Yoshioka T, Maeda S, Yonezawa S, Komiya S. Ribosomal protein S3 regulates GLI2-mediated osteosarcoma invasion. Cancer Lett 2014; 356:855-61. [PMID: 25449781 DOI: 10.1016/j.canlet.2014.10.042] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2014] [Revised: 10/29/2014] [Accepted: 10/30/2014] [Indexed: 11/16/2022]
Abstract
It has been reported that GLI2 promotes proliferation, migration, and invasion of mesenchymal stem cell and osteosarcoma cells. To examine the molecular mechanisms of GLI2-mediated osteosarcoma metastasis, we performed a microarray analysis. The gene encoding ribosomal protein S3 (RPS3) was identified as a target of GLI2. Real-time PCR revealed that RPS3 was upregulated in osteosarcoma cell lines compared with normal osteoblast cells. Knockdown of GLI2 decreased RPS3 expression, whereas forced expression of a constitutively active form of GLI2 upregulated the expression of RPS3. RPS3 knockdown by siRNA decreased the migration and invasion of osteosarcoma cells. Although forced expression of constitutively active GLI2 increased the migration of human mesenchymal stem cells, knockdown of RPS3 reduced the up-regulated migration. In contrast, forced expression of RPS3 increased migration and invasion of osteosarcoma cells. Moreover, reduction of migration by GLI2 knockdown was rescued by forced expression of RPS3. Immunohistochemical analysis showed that RPS3 expression was increased in primary osteosarcoma lesions with lung metastases compared with those without. These findings indicate that GLI2-RPS3 signaling may be a marker of invasive osteosarcoma and a therapeutic target for patients with osteosarcoma.
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Affiliation(s)
- Hiroko Nagao-Kitamoto
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Takao Setoguchi
- The Near-Future Locomotor Organ Medicine Creation Course (Kusunoki Kai), Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
| | - Sho Kitamoto
- Department of Human Pathology, Field of Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Shunsuke Nakamura
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Arisa Tsuru
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Masahito Nagata
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Satoshi Nagano
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Yasuhiro Ishidou
- Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Masahiro Yokouchi
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Shinichi Kitajima
- Department of Surgical Pathology, Kagoshima University Hospital, Kagoshima, Japan
| | - Takako Yoshioka
- Department of Molecular and Cellular Pathology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Shingo Maeda
- Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Suguru Yonezawa
- Department of Human Pathology, Field of Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
| | - Setsuro Komiya
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
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16
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Nagao-Kitamoto H, Nagata M, Nagano S, Kitamoto S, Ishidou Y, Yamamoto T, Nakamura S, Tsuru A, Abematsu M, Fujimoto Y, Yokouchi M, Kitajima S, Yoshioka T, Maeda S, Yonezawa S, Komiya S, Setoguchi T. GLI2 is a novel therapeutic target for metastasis of osteosarcoma. Int J Cancer 2014; 136:1276-84. [PMID: 25082385 DOI: 10.1002/ijc.29107] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Accepted: 06/27/2014] [Indexed: 11/05/2022]
Abstract
Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration-approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.
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Affiliation(s)
- Hiroko Nagao-Kitamoto
- Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
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Dai F, Yang S, Zhang F, Shi D, Zhang Z, Wu J, Xu J. hTERT- and hCTLA4Ig-expressing human bone marrow-derived mesenchymal stem cells: in vitro and in vivo characterization and osteogenic differentiation. J Tissue Eng Regen Med 2014; 11:400-411. [PMID: 25047146 DOI: 10.1002/term.1924] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Revised: 02/21/2014] [Accepted: 04/25/2014] [Indexed: 01/01/2023]
Abstract
Multipotent mesenchymal stem cells (MSCs) are commonly used as seed cells in studies of tissue engineering and regenerative medicine but their clinical application is limited, due to insufficient numbers of autogeneic MSCs, immune rejection of allogeneic MSCs and replicative senescence. We constructed two gene expression vectors for transfection of the human telomerase reverse transcriptase (hTERT) and cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4Ig) genes into human bone marrow-derived stem cells (hBMSCs). Successful transfection of both genes generated hTERT-CTLA4Ig hBMSCs that expressed both telomerase (shown by immunohistochemistry and a TRAPeze assay) and CTLA4Ig (demonstrated by immunocytochemistry and western blotting) without apparent mutual interference. Both hTERT BMSCs (92 population doublings) and hTERT-CTLA4Ig hBMSCs (60 population doublings) had an extended lifespan compared with hBMSCs (18 population doublings). Cell cycle analysis revealed that, compared with hBMSCs, a lower proportion of hTERT hBMSCs were in G0 /G1 phase but a higher proportion were in S phase; compared with hTERT hBMSCs, a higher proportion of hTERT-CTLA4Ig hBMSCs were in G0 /G1 phase, while a lower proportion were in S and G2 /M phases. hTERT-CTLA4Ig hBMSCs retained their capacity for osteogenic differentiation in vitro, shown by the detection of hydroxyapatite mineral deposition (labelled tetracycline fluorescence staining), calcareous nodules (alizarin red S staining), alkaline phosphatase (calcium-cobalt method) and osteocalcin (immunocytochemistry). Furthermore, subcutaneous transplantation of hTERT-CTLA4Ig hBMSCs in a rat xenotransplantation model resulted in the successful generation of bone-like tissue, confirmed using radiography and histological assessment. We propose that allogeneic hTERT-CTLA4Ig hBMSCs may be ideal seed cells for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.
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Affiliation(s)
- Fei Dai
- National and Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Sisi Yang
- Institute of Burns Research, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Fei Zhang
- National and Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Dongwen Shi
- Institute of Burns Research, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Zehua Zhang
- National and Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Jun Wu
- Institute of Burns Research, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
| | - Jianzhong Xu
- National and Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China
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Yamaguchi DT. “Ins” and “Outs” of mesenchymal stem cell osteogenesis in regenerative medicine. World J Stem Cells 2014; 6:94-110. [PMID: 24772237 PMCID: PMC3999785 DOI: 10.4252/wjsc.v6.i2.94] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/20/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
Repair and regeneration of bone requires mesenchymal stem cells that by self-renewal, are able to generate a critical mass of cells with the ability to differentiate into osteoblasts that can produce bone protein matrix (osteoid) and enable its mineralization. The number of human mesenchymal stem cells (hMSCs) diminishes with age and ex vivo replication of hMSCs has limited potential. While propagating hMSCs under hypoxic conditions may maintain their ability to self-renew, the strategy of using human telomerase reverse transcriptase (hTERT) to allow for hMSCs to prolong their replicative lifespan is an attractive means of ensuring a critical mass of cells with the potential to differentiate into various mesodermal structural tissues including bone. However, this strategy must be tempered by the oncogenic potential of TERT-transformed cells, or their ability to enhance already established cancers, the unknown differentiating potential of high population doubling hMSCs and the source of hMSCs (e.g., bone marrow, adipose-derived, muscle-derived, umbilical cord blood, etc.) that may provide peculiarities to self-renewal, differentiation, and physiologic function that may differ from non-transformed native cells. Tissue engineering approaches to use hMSCs to repair bone defects utilize the growth of hMSCs on three-dimensional scaffolds that can either be a base on which hMSCs can attach and grow or as a means of sequestering growth factors to assist in the chemoattraction and differentiation of native hMSCs. The use of whole native extracellular matrix (ECM) produced by hMSCs, rather than individual ECM components, appear to be advantageous in not only being utilized as a three-dimensional attachment base but also in appropriate orientation of cells and their differentiation through the growth factors that native ECM harbor or in simulating growth factor motifs. The origin of native ECM, whether from hMSCs from young or old individuals is a critical factor in “rejuvenating” hMSCs from older individuals grown on ECM from younger individuals.
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Jeon YM, Kook SH, Rho SJ, Lim SS, Choi KC, Kim HS, Kim JG, Lee JC. Fibroblast growth factor-7 facilitates osteogenic differentiation of embryonic stem cells through the activation of ERK/Runx2 signaling. Mol Cell Biochem 2013; 382:37-45. [PMID: 24026476 DOI: 10.1007/s11010-013-1716-5] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2013] [Accepted: 05/29/2013] [Indexed: 12/29/2022]
Abstract
Fibroblast growth factor-7 (FGF7) is known to regulate proliferation and differentiation of cells; however, little information is available on how FGF7 affects the differentiation of embryonic stem cells (ESCs). We examined the effects of FGF7 on proliferation and osteogenic differentiation of mouse ESCs. Exogenous FGF7 addition did not change the proliferation rate of mouse ESCs. In contrast, the addition of FGF7 facilitated the dexamethasone, ascorbic acid, and β-glycerophosphate (DAG)-induced increases in bone-like nodule formation and calcium accumulation. FGF7 also augmented mRNA expression of runt-related transcription factor-2 (Runx2), osterix, bone sialoprotein (BSP), and osteocalcin (OC) in the presence of DAG. FGF7-mediated increases in the mineralization and bone-specific gene expression were almost completely attenuated by pretreating with anti-FGF7 antibody. FGF7 treatment accelerated the DAG-induced activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the cells. A pharmacological inhibitor specific to ERK, but not to JNK or p38 kinase, dramatically suppressed FGF7-mediated mineralization and accumulation of collagen and OC in the presence of DAG. This suppression was accompanied by the reduction in Runx2, osterix, BSP, and OC mRNA levels, which were increased by FGF7 in the presence of DAG. Collectively, our results suggest that FGF7 stimulates osteogenic differentiation, but not proliferation, in ESCs, by activating ERK/Runx2 signaling.
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Affiliation(s)
- Young-Mi Jeon
- Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju, 561-756, South Korea
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Bischoff DS, Makhijani NS, Yamaguchi DT. Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells. Biores Open Access 2013; 1:273-9. [PMID: 23515239 PMCID: PMC3559209 DOI: 10.1089/biores.2012.0252] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.
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Affiliation(s)
- David S Bischoff
- Research Service, Veterans' Administration Greater Los Angeles Healthcare System , Los Angeles, California. ; The David Geffen School of Medicine at University of California , Los Angeles, California
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21
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Herlin C, Doucet JC, Bigorre M, Captier G. Computer-assisted midface reconstruction in Treacher Collins syndrome part 2: soft tissue reconstruction. J Craniomaxillofac Surg 2013; 41:676-80. [PMID: 23465636 DOI: 10.1016/j.jcms.2013.01.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2012] [Revised: 01/03/2013] [Accepted: 01/04/2013] [Indexed: 10/27/2022] Open
Abstract
INTRODUCTION Treacher Collins syndrome (TCS) midfacial involvement associate a skeletal hypoplasia centred on the zygoma to a hypoplasia of all surrounding soft tissues layers and an inferolateral lower lid pseudocoloboma. TCS soft tissue hypoplasia, which has not been well studied, continues to bring challenges in both the indication of surgical treatment and the prediction of their results. MATERIAL AND METHOD From a standard magnetic resonance imaging (MRI) acquisition, we studied qualitatively and quantitatively the prezygomatic fat compartments and the buccal fat pad of two individuals with TCS whose age were 10 and 14 years. In parallel, we studied 20 controls at the same age to obtain a morphometric database of reference and compare our results. TCS soft tissue involvement was correlated to the results of our prior skeletal involvement study. RESULTS The midfacial fat compartments in TCS are severely hypoplastic, especially in the superficial and lateral compartments of the face (all P's < 0.001). No significant correlation existed between the soft tissue and the skeletal involvement. CONCLUSIONS To our knowledge, this is the first published study of TCS midfacial fat compartments. Their hypoplasia is an important part of the syndrome's facial deformity. The knowledge of their anatomy, organization and volumetric variation is essential. Their re-establishment is key in the early treatment phases of this syndrome. Using the preoperative data, the morphometric database of reference, and surgical simulation, an appropriate surgical technique, going from an autologous fat graft to a free flap, can then be chose.
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Affiliation(s)
- Christian Herlin
- Department of Plastic and Craniofacial Pediatric Surgery (Head: Pr Guillaume Captier), Faculty of Medicine, University of Montpellier, 371 Avenue du doyen Gaston Giraud, 34295 Montpellier Cedex 5, France.
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Computer-assisted midface reconstruction in Treacher Collins syndrome part 1: skeletal reconstruction. J Craniomaxillofac Surg 2013; 41:670-5. [PMID: 23454266 DOI: 10.1016/j.jcms.2013.01.007] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2012] [Revised: 01/03/2013] [Accepted: 01/04/2013] [Indexed: 12/11/2022] Open
Abstract
INTRODUCTION Treacher Collins syndrome (TCS) is a severe and complex craniofacial malformation affecting the facial skeleton and soft tissues. The palate as well as the external and middle ear are also affected, but his prognosis is mainly related to neonatal airway management. Methods of zygomatico-orbital reconstruction are numerous and currently use primarily autologous bone, lyophilized cartilage, alloplastic implants, or even free flaps. This work developed a reliable "customized" method of zygomatico-orbital bony reconstruction using a generic reference model tailored to each patient. METHODS From a standard computed tomography (CT) acquisition, we studied qualitatively and quantitatively the skeleton of four individuals with TCS whose age was between 6 and 20 years. In parallel, we studied 40 controls at the same age to obtain a morphometric database of reference. Surgical simulation was carried out using validated software used in craniofacial surgery. RESULTS The zygomatic hypoplasia was very important quantitatively and morphologically in all TCS individuals. Orbital involvement was mainly morphological, with volumes comparable to the controls of the same age. The control database was used to create three-dimensional computer models to be used in the manufacture of cutting guides for autologous cranial bone grafts or alloplastic implants perfectly adapted to each patient's morphology. Presurgical simulation was also used to fabricate custom positioning guides permitting a simple and reliable surgical procedure. CONCLUSIONS The use of a virtual database allowed us to design a reliable and reproducible skeletal reconstruction method for this rare and complex syndrome. The use of presurgical simulation tools seem essential in this type of craniofacial malformation to increase the reliability of these uncommon and complex surgical procedures, and to ensure stable results over time.
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Piper SL, Wang M, Yamamoto A, Malek F, Luu A, Kuo AC, Kim HT. Inducible immortality in hTERT-human mesenchymal stem cells. J Orthop Res 2012; 30:1879-85. [PMID: 22674533 DOI: 10.1002/jor.22162] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Accepted: 05/09/2012] [Indexed: 02/04/2023]
Abstract
Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell-based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long-term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet-On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i-hTERT hMSCs). i-hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re-induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i-hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies.
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Affiliation(s)
- Samantha L Piper
- Department of Orthopaedic Surgery, University of California San Francisco, 500 Parnassus Avenue, San Francisco, California 94143, USA
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24
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Hayashi T, Misawa H, Nakahara H, Noguchi H, Yoshida A, Kobayashi N, Tanaka M, Ozaki T. Transplantation of osteogenically differentiated mouse iPS cells for bone repair. Cell Transplant 2012; 21:591-600. [PMID: 22793068 DOI: 10.3727/096368911x605529] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Induced pluripotent stem (iPS) cells are a type of undifferentiated cell that can be obtained from differentiated cells and have the pluripotent potential to differentiate into the musculoskeletal system, the myocardium, vascular endothelial cells, neurons, and hepatocytes. We therefore cultured mouse iPS cells in a DMEM containing 15% FBS, 10(-7) M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid for 3 weeks, in order to induce bone differentiation, and studied the expression of the bone differentiation markers Runx2 and osteocalcin using RT-PCR in a time-dependent manner. Osteocalcin, a bone differentiation marker in bone formation, exhibited the highest expression in the third week. In addition, the deposition of calcium nodules was observed using Alizarin red S staining. iPS cells cultured for bone differentiation were transplanted into severe combined immunodeficiency (SCID) mice, and the osteogenic potential exhibited after 4 weeks was studied. When bone differentiation-induced iPS cells were transplanted into SCID mice, bone formation was confirmed in soft X-ray images and tissue specimens. However, teratoma formation was confirmed in 20% of the transplanted models. When mouse iPS cells were treated with irradiation of 2 Gray (Gy) prior to transplantation, teratoma formation was inhibited. When mouse iPS cells treated in a likewise manner were xenotransplanted into rats, bone formation was confirmed but teratoma formation was not observed. It is believed that irradiation before transplantation is an effective way to inhibit teratoma formation.
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Affiliation(s)
- Takahiro Hayashi
- Department of Orthopeadic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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25
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Bone morphogenetic proteins in craniofacial surgery: current techniques, clinical experiences, and the future of personalized stem cell therapy. J Biomed Biotechnol 2012; 2012:601549. [PMID: 23226941 PMCID: PMC3511855 DOI: 10.1155/2012/601549] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2012] [Accepted: 10/16/2012] [Indexed: 12/29/2022] Open
Abstract
Critical-size osseous defects cannot heal without surgical intervention and can pose a significant challenge to craniofacial reconstruction. Autologous bone grafting is the gold standard for repair but is limited by a donor site morbidity and a potentially inadequate supply of autologous bone. Alternatives to autologous bone grafting include the use of alloplastic and allogenic materials, mesenchymal stem cells, and bone morphogenetic proteins. Bone morphogenetic proteins (BMPs) are essential mediators of bone formation involved in the regulation of differentiation of osteoprogenitor cells into osteoblasts. Here we focus on the use of BMPs in experimental models of craniofacial surgery and clinical applications of BMPs in the reconstruction of the cranial vault, palate, and mandible and suggest a model for the use of BMPs in personalized stem cell therapies.
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26
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Kang H, Sung J, Jung HM, Woo KM, Hong SD, Roh S. Insulin-Like Growth Factor 2 Promotes Osteogenic Cell Differentiation in the Parthenogenetic Murine Embryonic Stem Cells. Tissue Eng Part A 2012; 18:331-41. [DOI: 10.1089/ten.tea.2011.0074] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Affiliation(s)
- Hoin Kang
- Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul, Republic of Korea
| | - Jihye Sung
- Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul, Republic of Korea
| | - Hong-Moon Jung
- Department of Cell and Developmental Biology, Seoul National University School of Dentistry, Seoul, Republic of Korea
| | - Kyung Mi Woo
- Department of Cell and Developmental Biology, Seoul National University School of Dentistry, Seoul, Republic of Korea
| | - Seong-Doo Hong
- Department of Oral Pathology, Seoul National University School of Dentistry, Seoul, Republic of Korea
| | - Sangho Roh
- Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul, Republic of Korea
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27
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Jäger M, Hernigou P, Zilkens C, Herten M, Li X, Fischer J, Krauspe R. Cell therapy in bone healing disorders. Orthop Rev (Pavia) 2011; 2:e20. [PMID: 21808710 PMCID: PMC3143975 DOI: 10.4081/or.2010.e20] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2010] [Accepted: 08/19/2010] [Indexed: 12/20/2022] Open
Abstract
In addition to osteosynthetic stabilizing techniques and autologous bone transplantations, so-called orthobiologics play an increasing role in the treatment of bone healing disorders. Besides the use of various growth factors, more and more new data suggest that cell-based therapies promote local bone regeneration. For ethical and biological reasons, clinical application of progenitor cells on the musculoskeletal system is limited to autologous, postpartum stem cells. Intraoperative one-step treatment with autologous progenitor cells, in particular, delivered promising results in preliminary clinical studies. This article provides an overview of the rationale for, and characteristics of the clinical application of cell-based therapy to treat osseous defects based on a review of existing literature and our own experience with more than 100 patients. Most clinical trials report successful bone regeneration after the application of mixed cell populations from bone marrow. The autologous application of human bone marrow cells which are not expanded ex vivo has medico-legal advantages. However, there is a lack of prospective randomized studies including controls for cell therapy for bone defects. Autologous bone marrow cell therapy seems to be a promising treatment option which may reduce the amount of bone grafting in future.
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Affiliation(s)
- Marcus Jäger
- Dept. of Orthopaedics, University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Moorenstr Düsseldorf, Germany
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28
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Colnot C. Cell sources for bone tissue engineering: insights from basic science. TISSUE ENGINEERING PART B-REVIEWS 2011; 17:449-57. [PMID: 21902612 DOI: 10.1089/ten.teb.2011.0243] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
One of the goals of bone tissue engineering is to design delivery methods for skeletal stem/progenitor cells to repair or replace bone. Although the materials used to retain cells play a central role in the quality of the constructs, the source of cells is key for bone regeneration. Bone marrow is the most common cell source, but other tissues are now being explored, such as the periosteum, fat, muscle, cord blood, and embryonic or induced pluripotent stem cells. The therapeutic effect of exogenous stem/progenitor cells is accepted, yet their contribution to bone repair is not well defined. The in vitro osteo- and/or chondrogenic potential of these skeletal progenitors do not necessarily predict their differentiation potential in vivo and their function may be affected by their ability to home correctly to bone. This review provides an overview of animal models used to test the efficacy of cell-based approaches. We examine the mechanisms of endogenous cell recruitment during bone repair and compare the role of local versus systemic cell recruitment. We discuss how the normal repair process can help define efficacious cell sources for bone tissue engineering and improve their methods of delivery.
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Affiliation(s)
- Céline Colnot
- INSERM U781, Tour Lavoisier 2ème étage, Hôpital Necker-Enfants Malades, Paris, France.
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29
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Treatment of long bone defects and non-unions: from research to clinical practice. Cell Tissue Res 2011; 347:501-19. [PMID: 21574059 DOI: 10.1007/s00441-011-1184-8] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2011] [Accepted: 04/20/2011] [Indexed: 01/12/2023]
Abstract
The treatment of long bone defects and non-unions is still a major clinical and socio-economical problem. In addition to the non-operative therapeutic options, such as the application of various forms of electricity, extracorporeal shock wave therapy and ultrasound therapy, which are still in clinical use, several operative treatment methods are available. No consensus guidelines are available and the treatments of such defects differ greatly. Therefore, clinicians and researchers are presently investigating ways to treat large bone defects based on tissue engineering approaches. Tissue engineering strategies for bone regeneration seem to be a promising option in regenerative medicine. Several in vitro and in vivo studies in small and large animal models have been conducted to establish the efficiency of various tissue engineering approaches. Neverthelsss, the literature still lacks controlled studies that compare the different clinical treatment strategies currently in use. However, based on the results obtained so far in diverse animal studies, bone tissue engineering approaches need further validation in more clinically relevant animal models and in clinical pilot studies for the translation of bone tissue engineering approaches into clinical practice.
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30
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Nagao H, Ijiri K, Hirotsu M, Ishidou Y, Yamamoto T, Nagano S, Takizawa T, Nakashima K, Komiya S, Setoguchi T. Role of GLI2 in the growth of human osteosarcoma. J Pathol 2011; 224:169-79. [DOI: 10.1002/path.2880] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2010] [Revised: 02/12/2011] [Accepted: 02/20/2011] [Indexed: 12/20/2022]
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31
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Szpalski C, Barr J, Wetterau M, Saadeh PB, Warren SM. Cranial bone defects: current and future strategies. Neurosurg Focus 2010; 29:E8. [DOI: 10.3171/2010.9.focus10201] [Citation(s) in RCA: 147] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Bony defects in the craniomaxillofacial skeleton remain a major and challenging health concern. Surgeons have been trying for centuries to restore functionality and aesthetic appearance using autografts, allografts, and even xenografts without entirely satisfactory results. As a result, physicians, scientists, and engineers have been trying for the past few decades to develop new techniques to improve bone growth and bone healing. In this review, the authors summarize the advantages and limitations of current animal models; describe current materials used as scaffolds, cell-based, and protein-based therapies; and lastly highlight areas for future investigation. The purpose of this review is to highlight the major scaffold-, cell-, and protein-based preclinical tools that are currently being developed to repair cranial defects.
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32
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Daniels DJ, Clothier C, Swan DC, Saretzki G. Immediate and gradual gene expression changes in telomerase over-expressing fibroblasts. Biochem Biophys Res Commun 2010; 399:7-13. [DOI: 10.1016/j.bbrc.2010.07.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2010] [Accepted: 07/07/2010] [Indexed: 10/19/2022]
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33
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Jäger M, Hernigou P, Zilkens C, Herten M, Fischer J, Krauspe R. Zelltherapie bei Knochenheilungsstörungen. DER ORTHOPADE 2010; 39:449-62; quiz 463. [DOI: 10.1007/s00132-009-1583-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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34
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Shahid JM, Iwamuro M, Sasamoto H, Kubota Y, Seita M, Kawamoto H, Nakaji S, Noguchi H, Yamamoto K, Kobayashi N. Establishment of an immortalized porcine liver cell line JSNK-1 with retroviral transduction of SV40T. Cell Transplant 2010; 19:849-856. [PMID: 20955660 DOI: 10.3727/096368910x508979] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, β-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.
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Affiliation(s)
- Javed M Shahid
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
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