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Srila W, Pangjantuk A, Kunhorm P, Chaicharoenaudomrung N, Noisa P. Establishment and characterization of hTERT-immortalized porcine muscle stem cells, and their prospective uses. Food Sci Biotechnol 2025; 34:1597-1604. [PMID: 40129713 PMCID: PMC11929645 DOI: 10.1007/s10068-024-01785-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2024] [Revised: 11/12/2024] [Accepted: 11/27/2024] [Indexed: 03/26/2025] Open
Abstract
Myogenic differentiation ability made porcine muscle satellite cells (MSCs) promising candidates for cultured meat production. While in vitro, porcine primary cells lose differentiation capacity, have short lifespans, and change phenotype. For immortal porcine MSCs, human telomerase reverse transcriptase (hTERT) gene was overexpressed in parental cells to restore telomerase activity and lengthen cell longevity. After selection, G418-resistant cells were expanded and passed by different generations. The hTERT-immortalized MSCs presented spindle-like shape, telomere extension, and indefinite proliferation. In comparison to parental cells, immortal cells grew more rapidly and doubled faster. Immortal MSCs expressed muscle-specific protein and gene markers, were self-renewing stem cells, and could develop into myofibers in vitro. In culture plates with more than 100 generations, immortal MSCs formed tumors, but not lower passaged cells. Today, we showed that hTERT can immortalize primary porcine MSCs and preserve their stem cell characteristics. For research and cultured meat technologies, immortality may be valuable.
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Affiliation(s)
- Witsanu Srila
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand
- Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology, Thanyaburi, Pathumthani, 12110 Thailand
| | - Amorn Pangjantuk
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand
| | - Phongsakorn Kunhorm
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand
| | - Nipha Chaicharoenaudomrung
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand
| | - Parinya Noisa
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000 Thailand
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Bhardwaj R, Kumar L, Chhabra D, Sharma A, Mohanty S, Mehra N, Kochupillai V. Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity. Int J Hematol Oncol Stem Cell Res 2024; 18:330-343. [PMID: 39703476 PMCID: PMC11652691 DOI: 10.18502/ijhoscr.v18i4.16758] [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: 12/23/2023] [Accepted: 08/31/2024] [Indexed: 12/21/2024] Open
Abstract
Background: Human fetal liver hematopoietic stem cells have proven potential as therapeutics but lack extensive research due to their limited supply. Even in vitro expanded fetal liver hematopoietic stem cells enter senescence or lose their self-renewal capacity after a few days in culture. The present study aimed to obtain a homogeneous and persistent supply of hematopoietic stem cells from the fetal liver by establishing a cell line through immortalization of cells by enhancing telomerase activity. Materials and Methods: Human fetal liver hematopoietic CD34+ stem and progenitor cells were transformed and immortalized using retroviruses carrying the human telomerase (hTERT) gene. Following transduction, telomerase activity was assessed using the TRAP assay and telomere length was examined by Southern blotting in transduced cells. Their characterization was conducted using flowcytometry to analyze the CD34+ population of hematopoietic stem cells and their colony forming potential using colony forming unit (CFU) assay. Results: After transduction with hTERT, the life span of human fetal liver hematopoietic CD34+ stem and progenitor cells were extended to 80 population doublings, without any change in cell morphology or population doubling times. Constitutive hTERT expression enhanced the replicative capacity and prevented terminal differentiation of CD34+ fetal liver hematopoietic stem and progenitor cells (FLHSPCs). Moreover, hTERT-transduced stem cells maintained their telomere length and telomerase activity. Conclusion: By introducing telomerase activity into hematopoietic stem and progenitor cells, their lifespan can be extended while maintaining stemness. These modified cells hold promise for in vitro research focused on studying hematopoietic stem cells derived from fetal liver.
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Affiliation(s)
- Rashmi Bhardwaj
- Institute Rotary Cancer Hospital (IRCH), All India Institute of Medical Sciences (AIIMS), New Delhi, India
| | - Lalit Kumar
- Institute Rotary Cancer Hospital (IRCH), All India Institute of Medical Sciences (AIIMS), New Delhi, India
| | - Deepika Chhabra
- Sri Sri Institute for Advanced Research (SSIAR), Ved Vignan Maha Vidhya Peeth (VVMVP), Bangalore, India
| | - Atul Sharma
- Institute Rotary Cancer Hospital (IRCH), All India Institute of Medical Sciences (AIIMS), New Delhi, India
| | - Sujata Mohanty
- Stem Cell Facility, All India Institute of Medical Sciences (AIIMS), New Delhi, India
| | - Narinder Mehra
- Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences (AIIMS), New Delhi, India
| | - Vinod Kochupillai
- Sri Sri Institute for Advanced Research (SSIAR), Ved Vignan Maha Vidhya Peeth (VVMVP), Bangalore, India
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Shoeibi S, Green E, Wei H, Gou W, Strange C, Wang H. Immortalized mesenchymal stromal cells overexpressing alpha-1 antitrypsin protect acinar cells from apoptotic and ferroptotic cell death. J Cell Mol Med 2024; 28:e70093. [PMID: 39468387 PMCID: PMC11518823 DOI: 10.1111/jcmm.70093] [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: 12/01/2023] [Revised: 08/16/2024] [Accepted: 09/03/2024] [Indexed: 10/30/2024] Open
Abstract
Chronic pancreatitis (CP) is a progressive inflammatory disorder that impairs endocrine and exocrine function. Our previous work showed that mesenchymal stem/stromal cells (MSCs) and MSCs overexpressing alpha-1 antitrypsin (AAT-MSCs) could be therapeutic tools for CP. However, primary MSCs are predisposed to undergo senescence during culture expansion, which limits their therapeutic applications. We generated and characterized immortalized human MSCs (iMSCs) and AAT-MSCs (iAAT-MSCs) and tested their protective effect on 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced acinar cell death in an in vitro cell culture system. Primary MSCs were immortalized by transduction with simian virus 40 large T antigen (SV40LT), and the resulting iMSC and iAAT-MSC lines were analysed for proliferation, senescence, phenotype and multi-differentiation potential. Subsequently, apoptosis and ferroptosis pathways were investigated by assessing changes before and after TNBS treatment. Coculture of iMSCs and iAAT-MSCs with acinar cell lines inhibited early cell death induced by TNBS, reduced ER stress and reversed TNBS-induced protein reduction at tight junctions. Additionally, iMSCs and iAAT-MSCs exerted such protection by regulating mitochondrial respiration, ATP content and ROS production in TNBS-induced acinar cells. Furthermore, iMSCs and iAAT-MSCs ameliorated TNBS-induced ferroptosis by modulating iron generation and ROS production and regulating the ferritin heavy chain 1 (FTH1)/protein disulfide isomerase (PDI)/glutathione peroxide 4 (GPX4) signalling pathways in acinar cells. These findings identify ferroptosis as an unrecognized mechanism that leads to TNBS-induced cell death and offer mechanistic insights relevant to using stem cell therapy to treat acinar cell death associated with CP.
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Affiliation(s)
- Sara Shoeibi
- Department of SurgeryMedical University of South CarolinaCharlestonSouth CarolinaUSA
| | - Erica Green
- Department of SurgeryMedical University of South CarolinaCharlestonSouth CarolinaUSA
| | - Hua Wei
- Department of SurgeryMedical University of South CarolinaCharlestonSouth CarolinaUSA
| | - Wenyu Gou
- Department of SurgeryMedical University of South CarolinaCharlestonSouth CarolinaUSA
| | - Charlie Strange
- Department of MedicineMedical University of South CarolinaCharlestonSouth CarolinaUSA
| | - Hongjun Wang
- Department of SurgeryMedical University of South CarolinaCharlestonSouth CarolinaUSA
- Ralph H. Johnson Veterans Affairs Medical CenterCharlestonSouth CarolinaUSA
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Colella A, Biondi G, Marrano N, Francioso E, Fracassi L, Crovace AM, Recchia A, Natalicchio A, Paradies P. Generation of Insulin-Producing Cells from Canine Bone Marrow-Derived Mesenchymal Stem Cells: A Preliminary Study. Vet Sci 2024; 11:380. [PMID: 39195834 PMCID: PMC11359947 DOI: 10.3390/vetsci11080380] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 08/07/2024] [Accepted: 08/13/2024] [Indexed: 08/29/2024] Open
Abstract
Cell-based therapy using insulin-producing cells (IPCs) is anticipated as an alternative treatment option to insulin injection or pancreatic islet transplantation for the treatment of diabetes mellitus in both human and veterinary medicine. Several protocols were reported for the differentiation of mesenchymal stem cells (MSCs) into IPCs; to date, glucose-responsive IPCs have only been obtained from canine adipose tissue-derived MSCs (cAD-MSCs), but not from canine bone marrow-derived MSCs (cBM-MSCs). Therefore, this study aims to generate in vitro glucose-responsive IPCs from cBM-MSCs using two differentiation protocols: a two-step protocol using trichostatin (TSA) and a three-step protocol using mercaptoethanol to induce pancreatic and duodenal homeobox gene 1 (PDX-1) expression. A single experiment was carried out for each protocol. BM-MSCs from one dog were successfully cultured and expanded. Cells exposed to the two-step protocol appeared rarely grouped to form small clusters; gene expression analysis showed a slight increase in PDX-1 and insulin expression, but no insulin protein production nor secretion in the culture medium was detected either under basal conditions or following glucose stimulation. Conversely, cells exposed to the three-step protocol under a 3D culture system formed colony-like structures; insulin gene expression was upregulated compared to undifferentiated control and IPCs colonies secreted insulin in the culture medium, although insulin secretion was not enhanced by high-glucose culture conditions. The single experiment results suggest that the three-step differentiation protocol could generate IPCs from cBM-MSCs; however, further experiments are needed to confirm these data. The ability of IPCs from cBM- MSCs to produce insulin, described here for the first time, is a preliminary interesting result. Nevertheless, the IPCs' unresponsiveness to glucose, if confirmed, would affect its clinical application. Further studies are necessary to establish a differentiation protocol in this perspective.
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Affiliation(s)
- Antonella Colella
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Veterinary Clinics and Animal Production Section, University of Bari Aldo Moro, Valenzano, 70010 Bari, Italy; (A.C.); (E.F.); (L.F.); (A.R.)
| | - Giuseppina Biondi
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Internal Medicine, Endocrinology, Andrology and Metabolic Diseases Section, University of Bari Aldo Moro, 70124 Bari, Italy; (G.B.); (N.M.); (A.N.)
| | - Nicola Marrano
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Internal Medicine, Endocrinology, Andrology and Metabolic Diseases Section, University of Bari Aldo Moro, 70124 Bari, Italy; (G.B.); (N.M.); (A.N.)
| | - Edda Francioso
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Veterinary Clinics and Animal Production Section, University of Bari Aldo Moro, Valenzano, 70010 Bari, Italy; (A.C.); (E.F.); (L.F.); (A.R.)
| | - Laura Fracassi
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Veterinary Clinics and Animal Production Section, University of Bari Aldo Moro, Valenzano, 70010 Bari, Italy; (A.C.); (E.F.); (L.F.); (A.R.)
| | - Alberto M. Crovace
- Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy;
| | - Alessandra Recchia
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Veterinary Clinics and Animal Production Section, University of Bari Aldo Moro, Valenzano, 70010 Bari, Italy; (A.C.); (E.F.); (L.F.); (A.R.)
| | - Annalisa Natalicchio
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Internal Medicine, Endocrinology, Andrology and Metabolic Diseases Section, University of Bari Aldo Moro, 70124 Bari, Italy; (G.B.); (N.M.); (A.N.)
| | - Paola Paradies
- Department of Precision and Regenerative Medicine and Ionian Area (DiMePre-J), Veterinary Clinics and Animal Production Section, University of Bari Aldo Moro, Valenzano, 70010 Bari, Italy; (A.C.); (E.F.); (L.F.); (A.R.)
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Hindle J, Williams A, Kim Y, Kim D, Patil K, Khatkar P, Osgood Q, Nelson C, Routenberg DA, Howard M, Liotta LA, Kashanchi F, Branscome H. hTERT-Immortalized Mesenchymal Stem Cell-Derived Extracellular Vesicles: Large-Scale Manufacturing, Cargo Profiling, and Functional Effects in Retinal Epithelial Cells. Cells 2024; 13:861. [PMID: 38786083 PMCID: PMC11120263 DOI: 10.3390/cells13100861] [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: 04/13/2024] [Revised: 05/07/2024] [Accepted: 05/14/2024] [Indexed: 05/25/2024] Open
Abstract
As the economic burden associated with vision loss and ocular damage continues to rise, there is a need to explore novel treatment strategies. Extracellular vesicles (EVs) are enriched with various biological cargo, and there is abundant literature supporting the reparative and immunomodulatory properties of stem cell EVs across a broad range of pathologies. However, one area that requires further attention is the reparative effects of stem cell EVs in the context of ocular damage. Additionally, most of the literature focuses on EVs isolated from primary stem cells; the use of EVs isolated from human telomerase reverse transcriptase (hTERT)-immortalized stem cells has not been thoroughly examined. Using our large-scale EV-manufacturing platform, we reproducibly manufactured EVs from hTERT-immortalized mesenchymal stem cells (MSCs) and employed various methods to characterize and profile their associated cargo. We also utilized well-established cell-based assays to compare the effects of these EVs on both healthy and damaged retinal pigment epithelial cells. To the best of our knowledge, this is the first study to establish proof of concept for reproducible, large-scale manufacturing of hTERT-immortalized MSC EVs and to investigate their potential reparative properties against damaged retinal cells. The results from our studies confirm that hTERT-immortalized MSC EVs exert reparative effects in vitro that are similar to those observed in primary MSC EVs. Therefore, hTERT-immortalized MSCs may represent a more consistent and reproducible platform than primary MSCs for generating EVs with therapeutic potential.
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Affiliation(s)
| | - Anastasia Williams
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Yuriy Kim
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | | | - Kajal Patil
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Pooja Khatkar
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | | | - Collin Nelson
- Meso Scale Diagnostics, L.L.C., Rockville, MD 20850, USA (D.A.R.)
| | | | - Marissa Howard
- Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA 20110, USA
| | - Lance A. Liotta
- Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA 20110, USA
| | - Fatah Kashanchi
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Heather Branscome
- ATCC, Manassas, VA 20110, USA
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
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Musgrove L, Russell FD, Ventura T. Considerations for cultivated crustacean meat: potential cell sources, potential differentiation and immortalization strategies, and lessons from crustacean and other animal models. Crit Rev Food Sci Nutr 2024; 65:2431-2455. [PMID: 38733287 DOI: 10.1080/10408398.2024.2342480] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/13/2024]
Abstract
Cultivated crustacean meat (CCM) is a means to create highly valued shrimp, lobster, and crab products directly from stem cells, thus removing the need to farm or fish live animals. Conventional crustacean enterprises face increasing pressures in managing overfishing, pollution, and the warming climate, so CCM may provide a way to ensure sufficient supply as global demand for these products grows. To support the development of CCM, this review briefly details crustacean cell culture work to date, before addressing what is presently known about crustacean muscle development, particularly the molecular mechanisms involved, and how this might relate to recent work on cultivated meat production in vertebrate species. Recognizing the current lack of cell lines available to establish CCM cultures, we also consider primary stem cell sources that can be obtained non-lethally including tissues from limbs which are readily released and regrown, and putative stem cells in circulating hemolymph. Molecular approaches to inducing myogenic differentiation and immortalization of putative stem cells are also reviewed. Finally, we assess the current status of tools available to CCM researchers, particularly antibodies, and propose avenues to address existing shortfalls in order to see the field progress.
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Affiliation(s)
- Lisa Musgrove
- Centre for Bioinnovation, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
- School of Science, Technology and Engineering, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
| | - Fraser D Russell
- Centre for Bioinnovation, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
- School of Health, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
| | - Tomer Ventura
- Centre for Bioinnovation, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
- School of Science, Technology and Engineering, University of the Sunshine Coast (UniSC), Maroochydore, QLD, Australia
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Rajput SN, Naeem BK, Ali A, Salim A, Khan I. Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine. World J Stem Cells 2024; 16:410-433. [PMID: 38690517 PMCID: PMC11056638 DOI: 10.4252/wjsc.v16.i4.410] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 02/01/2024] [Accepted: 03/18/2024] [Indexed: 04/25/2024] Open
Abstract
BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. METHODS Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. RESULTS Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.
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Affiliation(s)
- Shafiqa Naeem Rajput
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Bushra Kiran Naeem
- Surgical Unit 4, Dr. Ruth KM Pfau Civil Hospital, Karachi 74400, Pakistan
| | - Anwar Ali
- Department of Physiology, University of Karachi, Karachi 75270, Pakistan
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
- Center for Regenerative Medicine and Stem Cells Research, and Department of Ophthalmology and Visual Sciences, The Aga Khan University, Karachi 74800, Sindh, Pakistan.
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Stricker PEF, de Oliveira NB, Mogharbel BF, Lührs L, Irioda AC, Abdelwahid E, Regina Cavalli L, Zotarelli-Filho IJ, de Carvalho KAT. Meta-analysis of the Mesenchymal Stem Cells Immortalization Protocols: A Guideline for Regenerative Medicine. Curr Stem Cell Res Ther 2024; 19:1009-1020. [PMID: 38221663 DOI: 10.2174/011574888x268464231016070900] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2023] [Revised: 08/28/2023] [Accepted: 09/11/2023] [Indexed: 01/16/2024]
Abstract
BACKGROUND This systematic review describes the most common methodologies for immortalizing human and animal mesenchymal stem cells (MSCs). This study follows the rules of PRISMA and is registered in the Institutional Review Board of PROSPERO International of systematic reviews, numbered protocol code: CRD42020202465. METHOD The data search systematization was based on the words "mesenchymal stem cell" AND "immortalization." The search period for publications was between 2000 and 2022, and the databases used were SCOPUS, PUBMED, and SCIENCE DIRECT. The search strategies identified 384 articles: 229 in the SCOPUS database, 84 in PUBMED, and 71 in SCIENCE DIRECT. After screening by titles and abstracts, 285 articles remained. This review included thirty-nine articles according to the inclusion and exclusion criteria. RESULT In 28 articles, MSCs were immortalized from humans and 11 animals. The most used immortalization methodology was viral transfection. The most common immortalized cell type was the MSC from bone marrow, and the most used gene for immortalizing human and animal MSCs was hTERT (39.3%) and SV40T (54.5%), respectively. CONCLUSION Also, it was observed that although less than half of the studies performed tumorigenicity assays to validate the immortalized MSCs, other assays, such as qRT-PCR, colony formation in soft agar, karyotype, FISH, and cell proliferation, were performed in most studies on distinct MSC cell passages.
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Affiliation(s)
| | | | - Bassam Felipe Mogharbel
- Pelé Pequeno Príncipe Research Institute Research & Pequeno Príncipe Faculties, Curitiba, Brazil
| | - Larissa Lührs
- Pelé Pequeno Príncipe Research Institute Research & Pequeno Príncipe Faculties, Curitiba, Brazil
| | - Ana Carolina Irioda
- Pelé Pequeno Príncipe Research Institute Research & Pequeno Príncipe Faculties, Curitiba, Brazil
| | - Eltyeb Abdelwahid
- Feinberg School of Medicine, Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL, USA
| | - Luciane Regina Cavalli
- Pelé Pequeno Príncipe Research Institute Research & Pequeno Príncipe Faculties, Curitiba, Brazil
| | - Idiberto José Zotarelli-Filho
- Pelé Pequeno Príncipe Research Institute Research & Pequeno Príncipe Faculties, Curitiba, Brazil
- ABRAN - Associação Brasileira de Nutrologia/Brazilian Association of Nutrology, Catanduva, Sao Paulo, Brazil
- College of Palliative Medicine of Sri Lanka, Colombo, Sri Lanka
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Rakhmatullina AR, Mingaleeva RN, Gafurbaeva DU, Glazunova ON, Sagdeeva AR, Bulatov ER, Rizvanov AA, Miftakhova RR. Adipose-Derived Mesenchymal Stem Cell (MSC) Immortalization by Modulation of hTERT and TP53 Expression Levels. J Pers Med 2023; 13:1621. [PMID: 38003936 PMCID: PMC10672200 DOI: 10.3390/jpm13111621] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2023] [Revised: 11/03/2023] [Accepted: 11/13/2023] [Indexed: 11/26/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are pivotal players in tissue repair and hold great promise as cell therapeutic agents for regenerative medicine. Additionally, they play a significant role in the development of various human diseases. Studies on MSC biology have encountered a limiting property of these cells, which includes a low number of passages and a decrease in differentiation potential during in vitro culture. Although common methods of immortalization through gene manipulations of cells are well established, the resulting MSCs vary in differentiation potential compared to primary cells and eventually undergo senescence. This study aimed to immortalize primary adipose-derived MSCs by overexpressing human telomerase reverse transcriptase (hTERT) gene combined with a knockdown of TP53. The research demonstrated that immortalized MSCs maintained a stable level of differentiation into osteogenic and chondrogenic lineages during 30 passages, while also exhibiting an increase in cell proliferation rate and differentiation potential towards the adipogenic lineage. Long-term culture of immortalized cells did not alter cell morphology and self-renewal potential. Consequently, a genetically stable line of immortalized adipose-derived MSCs (iMSCs) was established.
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Affiliation(s)
- Aigul R. Rakhmatullina
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Rimma N. Mingaleeva
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Dina U. Gafurbaeva
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Olesya N. Glazunova
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Aisylu R. Sagdeeva
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Emil R. Bulatov
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
| | - Albert A. Rizvanov
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
| | - Regina R. Miftakhova
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
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10
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Liu TM, Wu Y. Editorial: Advancing gene and cell therapy using human mesenchymal stem cells. Front Cell Dev Biol 2023; 11:1294460. [PMID: 37900271 PMCID: PMC10613041 DOI: 10.3389/fcell.2023.1294460] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2023] [Accepted: 10/09/2023] [Indexed: 10/31/2023] Open
Affiliation(s)
- Tong Ming Liu
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
| | - Yingnan Wu
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore, Singapore
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11
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Wang X, Ouyang L, Chen W, Cao Y, Zhang L. Efficient expansion and delayed senescence of hUC-MSCs by microcarrier-bioreactor system. Stem Cell Res Ther 2023; 14:284. [PMID: 37794520 PMCID: PMC10552362 DOI: 10.1186/s13287-023-03514-1] [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: 06/06/2023] [Accepted: 09/25/2023] [Indexed: 10/06/2023] Open
Abstract
BACKGROUND Human umbilical cord mesenchymal stem cells (hUC-MSCs) are widely used in cell therapy due to their robust immunomodulatory and tissue regenerative capabilities. Currently, the predominant method for obtaining hUC-MSCs for clinical use is through planar culture expansion, which presents several limitations. Specifically, continuous cell passaging can lead to cellular aging, susceptibility to contamination, and an absence of process monitoring and control, among other limitations. To overcome these challenges, the technology of microcarrier-bioreactor culture was developed with the aim of ensuring the therapeutic efficacy of cells while enabling large-scale expansion to meet clinical requirements. However, there is still a knowledge gap regarding the comparison of biological differences in cells obtained through different culture methods. METHODS We developed a culture process for hUC-MSCs using self-made microcarrier and stirred bioreactor. This study systematically compares the biological properties of hUC-MSCs amplified through planar culture and microcarrier-bioreactor systems. Additionally, RNA-seq was employed to compare the differences in gene expression profiles between the two cultures, facilitating the identification of pathways and genes associated with cell aging. RESULTS The findings revealed that hUC-MSCs expanded on microcarriers exhibited a lower degree of cellular aging compared to those expanded through planar culture. Additionally, these microcarrier-expanded hUC-MSCs showed an enhanced proliferation capacity and a reduced number of cells in the cell cycle retardation period. Moreover, bioreactor-cultured cells differ significantly from planar cultures in the expression of genes associated with the cytoskeleton and extracellular matrix. CONCLUSIONS The results of this study demonstrate that our microcarrier-bioreactor culture method enhances the proliferation efficiency of hUC-MSCs. Moreover, this culture method exhibits the potential to delay the process of cell aging while preserving the essential stem cell properties of hUC-MSCs.
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Affiliation(s)
- Xia Wang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
| | - Liming Ouyang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
| | - Wenxia Chen
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
| | - Yulin Cao
- Beijing Tang Yi Hui Kang Biomedical Technology Co., LTD, Beijing, 100032, People's Republic of China
| | - Lixin Zhang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
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12
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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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13
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Shoeibi S, Green E, Wei H, Gou W, Strange C, Wang H. Immortalized Mesenchymal Stromal Cells Overexpressing Alpha-1 Antitrypsin Protect Acinar Cells from Apoptotic and Ferroptotic Cell Death. RESEARCH SQUARE 2023:rs.3.rs-2961444. [PMID: 37609340 PMCID: PMC10441457 DOI: 10.21203/rs.3.rs-2961444/v1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/24/2023]
Abstract
Chronic pancreatitis (CP) is a progressive inflammatory disorder that impairs endocrine and exocrine function. Our previous work suggests that mesenchymal stem/stromal cells (MSCs) and MSCs overexpressing alpha-1 antitrypsin (AAT-MSCs) could be therapeutic tools for CP treatment in mouse models. However, primary MSCs have a predisposition to undergo senescence during culture expansion which limits their therapeutic applications. Here we generated and characterized immortalized human MSCs (iMSCs) and AAT-MSCs (iAAT-MSCs) and tested their protective effect on 2,4,6-Trinitrobenzenesulfonic acid (TNBS) -induced acinar cell death in an in vitro cell culture system. Primary MSCs were immortalized by transduction with simian virus 40 large T antigen (SV40LT), and the resulting iMSC and iAAT-MSC lines were analyzed for proliferation, senescence, phenotype, and multi-differentiation potential. Subsequently, the impact of these cells on TNBS-induced cell death was measured and compared. Both apoptosis and ferroptosis pathways were investigated by assessing changes of critical factors before and after cell treatment. Coculture of iMSCs and iAAT-MSCs with acinar cell lines inhibited early apoptosis induced by TNBS, reduced ER stress, and reversed TNBS-induced protein reduction at tight junctions. Additionally, iMSCs and iAAT-MSCs exerted such protection by regulating mitochondrial respiration, ATP content, and ROS production in TNBS-induced acinar cells. Furthermore, iMSCs and iAAT-MSCs ameliorated ferroptosis by regulating the ferritin heavy chain 1 (FTH1)/protein disulfide isomerase (PDI)/glutathione peroxide 4 (GPX4) signaling pathways and by modulating ROS function and iron generation in acinar cells. These findings identified ferroptosis as one of the mechanisms that leads to TNBS-induced cell death and offer mechanistic insights relevant to using stem cell therapy for the treatment of CP.
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Affiliation(s)
| | | | | | - Wenyu Gou
- Medical University of South Carolina
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14
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Lee SS, Vũ TT, Weiss AS, Yeo GC. Stress-induced senescence in mesenchymal stem cells: Triggers, hallmarks, and current rejuvenation approaches. Eur J Cell Biol 2023; 102:151331. [PMID: 37311287 DOI: 10.1016/j.ejcb.2023.151331] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 06/04/2023] [Accepted: 06/05/2023] [Indexed: 06/15/2023] Open
Abstract
Mesenchymal stem cells (MSCs) have emerged as promising cell-based therapies in the treatment of degenerative and inflammatory conditions. However, despite accumulating evidence of the breadth of MSC functional potency, their broad clinical translation is hampered by inconsistencies in therapeutic efficacy, which is at least partly due to the phenotypic and functional heterogeneity of MSC populations as they progress towards senescence in vitro. MSC senescence, a natural response to aging and stress, gives rise to altered cellular responses and functional decline. This review describes the key regenerative properties of MSCs; summarises the main triggers, mechanisms, and consequences of MSC senescence; and discusses current cellular and extracellular strategies to delay the onset or progression of senescence, or to rejuvenate biological functions lost to senescence.
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Affiliation(s)
- Sunny Shinchen Lee
- Charles Perkins Centre, The University of Sydney, NSW 2006, Australia; School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia
| | - Thu Thuy Vũ
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Viet Nam
| | - Anthony S Weiss
- Charles Perkins Centre, The University of Sydney, NSW 2006, Australia; School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia; Sydney Nano Institute, The University of Sydney, NSW 2006, Australia
| | - Giselle C Yeo
- Charles Perkins Centre, The University of Sydney, NSW 2006, Australia; School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia.
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15
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Sutyagina OI, Beilin AK, Vorotelyak EA, Vasiliev AV. Immortalization Reversibility in the Context of Cell Therapy Biosafety. Int J Mol Sci 2023; 24:7738. [PMID: 37175444 PMCID: PMC10178325 DOI: 10.3390/ijms24097738] [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: 03/09/2023] [Revised: 04/18/2023] [Accepted: 04/20/2023] [Indexed: 05/15/2023] Open
Abstract
Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.
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Affiliation(s)
- Oksana I. Sutyagina
- N.K. Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Laboratory of Cell Biology, Vavilov Str. 26, 119334 Moscow, Russia
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16
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Sanz-Serrano D, Sánchez-de-Diego C, Mercade M, Ventura F. Dental Stem Cells SV40, a new cell line developed in vitro from human stem cells of the apical papilla. Int Endod J 2023; 56:502-513. [PMID: 36585930 DOI: 10.1111/iej.13887] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2021] [Revised: 12/24/2022] [Accepted: 12/26/2022] [Indexed: 01/01/2023]
Abstract
AIM To establish and fully characterize a new cell line from human stem cells of the apical papilla (SCAPs) through immortalization with an SV40 large T antigen. METHODOLOGY Human SCAPs were isolated and transfected with an SV40 large T antigen and treated with puromycin to select the infected population. Expression of human mesenchymal surface markers CD73, CD90 and CD105 was assessed in the new cell line named Dental Stem Cells SV40 (DSCS) by flow cytometry at early and late passages. Cell contact inhibition and proliferation were also analysed. To evaluate trilineage differentiation, quantitative polymerase chain reaction and histological staining were performed. RESULTS DSCS cell flow cytometry confirmed the expression of mesenchymal surface markers even in late passages [100% positive for CD73 and CD90 and 98.9% for CD105 at passage (P) 25]. Fewer than 0.5% were positive for haematopoietic cell markers (CD45 and CD34). DSCS cells also showed increased proliferation when compared to the primary culture after 48 h, with a doubling time of 23.46 h for DSCS cells and 40.31 h for SCAPs, and retained the capacity to grow for >45 passages (150 population doubling) and their spindle-shaped morphology. Trilineage differentiation potential was confirmed through histochemical staining and gene expression of the chondrogenic markers SOX9 and COL2A1, adipogenic markers CEBPA and LPL, and osteogenic markers COL1A1 and ALPL. CONCLUSIONS The new cell line derived from human SCAPs has multipotency, retains its morphology and expression of mesenchymal surface markers and shows higher proliferative capacity even at late passages (P45). DSCS cells can be used for in vitro study of root development and to achieve a better understanding of the regenerative mechanisms.
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Affiliation(s)
- Diana Sanz-Serrano
- Department of Dentistry, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain
| | - Cristina Sánchez-de-Diego
- Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA.,University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA
| | - Montse Mercade
- Department of Dentistry, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, Spain.,Researcher at IDIBELL Institute, L'Hospitalet de Llobregat, Spain
| | - Francesc Ventura
- Researcher at IDIBELL Institute, L'Hospitalet de Llobregat, Spain.,Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, Spain
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17
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CRISPR/Cas9-engineered mesenchymal stromal/stem cells and their extracellular vesicles: A new approach to overcoming cell therapy limitations. Biomed Pharmacother 2022; 156:113943. [DOI: 10.1016/j.biopha.2022.113943] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2022] [Revised: 10/21/2022] [Accepted: 10/26/2022] [Indexed: 11/11/2022] Open
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18
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Rizvi SFA, Wasim B, Usman S, Borges KJJ, Sahibdad I, Salim A, Khan I. Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells. Mol Cell Biochem 2022; 477:2735-2749. [PMID: 35610401 DOI: 10.1007/s11010-022-04468-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Accepted: 05/04/2022] [Indexed: 11/09/2022]
Abstract
The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.
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Affiliation(s)
- Syed Faizan Ali Rizvi
- Ghulam Muhammad Mahar Medical College Sukkur at Shaheed Mohtarma Benazir Bhutto Medical University Larkana, Larkana, 77150, Pakistan
- Ziauddin University, Clifton, Karachi, 74700, Pakistan
| | - Bushra Wasim
- Ziauddin University, Clifton, Karachi, 74700, Pakistan
| | | | | | - Iqra Sahibdad
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Centre for Chemical and Biological Sciences, Karachi, 75270, Pakistan
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Centre for Chemical and Biological Sciences, Karachi, 75270, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Centre for Chemical and Biological Sciences, Karachi, 75270, Pakistan.
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.
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19
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Dos Santos A, Lyu N, Balayan A, Knight R, Zhuo KS, Sun Y, Xu J, Funderburgh ML, Funderburgh JL, Deng SX. Generation of Functional Immortalized Human Corneal Stromal Stem Cells. Int J Mol Sci 2022; 23:13399. [PMID: 36362184 PMCID: PMC9657819 DOI: 10.3390/ijms232113399] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 10/26/2022] [Accepted: 10/28/2022] [Indexed: 06/04/2024] Open
Abstract
In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.
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Affiliation(s)
- Aurelie Dos Santos
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Ning Lyu
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
- Department of Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai 200031, China
| | - Alis Balayan
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Rob Knight
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Katherine Sun Zhuo
- Human Biology Society, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Yuzhao Sun
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
- Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China
| | - Jianjiang Xu
- Department of Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai 200031, China
| | | | | | - Sophie X. Deng
- Stein Eye Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
- Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
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20
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Iacomi DM, Rosca AM, Tutuianu R, Neagu TP, Pruna V, Simionescu M, Titorencu I. Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy. Int J Mol Sci 2022; 23:ijms23168925. [PMID: 36012192 PMCID: PMC9408591 DOI: 10.3390/ijms23168925] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 07/29/2022] [Accepted: 08/06/2022] [Indexed: 11/25/2022] Open
Abstract
Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells’ lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.
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Affiliation(s)
- Daniela-Madalina Iacomi
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Ana-Maria Rosca
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
- Correspondence:
| | - Raluca Tutuianu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Tiberiu Paul Neagu
- Clinical Department No. 11, “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Vasile Pruna
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Maya Simionescu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Irina Titorencu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
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21
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Knight R, Board-Davies E, Brown H, Clayton A, Davis T, Karatas B, Burston J, Tabi Z, Falcon-Perez JM, Paisey S, Stephens P. Oral Progenitor Cell Line-Derived Small Extracellular Vesicles as a Treatment for Preferential Wound Healing Outcome. Stem Cells Transl Med 2022; 11:861-875. [PMID: 35716044 PMCID: PMC9397654 DOI: 10.1093/stcltm/szac037] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Accepted: 04/28/2022] [Indexed: 12/11/2022] Open
Abstract
Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.
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Affiliation(s)
- Rob Knight
- Regenerative Biology Group, Oral and Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff, Wales, UK,Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK,PETIC, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Emma Board-Davies
- Regenerative Biology Group, Oral and Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff, Wales, UK,Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK
| | - Helen Brown
- Regenerative Biology Group, Oral and Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff, Wales, UK,Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK
| | - Aled Clayton
- Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK,Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Terence Davis
- PETIC, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Ben Karatas
- Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK,Systems Immunity Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - James Burston
- Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK,Systems Immunity Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK,Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Zsuzsanna Tabi
- PETIC, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Juan M Falcon-Perez
- Exosomes Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain,Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain,IKERBASQUE, Basque Foundation for Science, Bilbao, Bizkaia, Spain
| | - Stephen Paisey
- Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, Wales, UK,PETIC, School of Medicine, Cardiff University, Cardiff, Wales, UK
| | - Phil Stephens
- Corresponding author: Phil Stephens, Regenerative Biology Group, Oral and Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff, CF14 4XY, Wales, UK.
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22
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Wang Y, Pei YA, Sun Y, Zhou S, Zhang XB, Pei M. Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation, differentiation, and reconstruction of matrix microenvironment. Acta Biomater 2021; 136:184-198. [PMID: 34551328 PMCID: PMC8627502 DOI: 10.1016/j.actbio.2021.09.021] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 09/13/2021] [Accepted: 09/14/2021] [Indexed: 11/22/2022]
Abstract
Although matrix microenvironment has the potential to improve expanded stem cell proliferation and differentiation capacity, decellularized extracellular matrix (dECM) deposited by senescent cells does not contribute to the rejuvenation of adult stem cells, which has become a barrier to personalized stem cell therapy. Genetic modification is an effective strategy to protect cells from senescence but it carries the increased risk of malignant transformation and genetic instability. In this study, lentivirus carrying either human telomerase reverse transcriptase (hTERT) or simian virus 40 large T antigen (SV40LT) was used to transduce human infrapatellar fat pad-derived stem cells (IPFSCs). We found that virus transduction modified the proliferative, chondrogenic, and adipogenic abilities of IPFSCs. Interestingly, dECM deposited by immortalized cells significantly influenced replicative senescent IPFSCs in proliferation and differentiation preference, the effect of which is hinged on the approach of immortalization using either SV40LT or hTERT. Our findings indicate both dECM expansion and immortalization strategies can be used for replicative senescent adult stem cells' proliferation and lineage-specific differentiation, which benefits future stem cell-based tissue regeneration. This approach may also work for adult stem cells with premature senescence in elderly/aged patients, which needs further investigation. STATEMENT OF SIGNIFICANCE: Adult stem cells are a promising solution for autologous cell-based therapy. Unfortunately, cell senescence due to donor age and/or ex vivo expansion prevents clinical application. Recent progress with decellularized extracellular matrix provides a potential for the rejuvenation of senescent stem cells by improving their proliferation and differentiation capacities. Given the fact that the young matrix can provide a healthy and energetic microenvironment, in this study, two approaches using lentivirus transduction of hTERT and SV40LT were compared. The goal was to immortalize donor cells for deposition of decellularized extracellular matrix. The matrix was demonstrated to contribute diverging effects on the chondrogenic and adipogenic differentiation of expanded stem cells and exhibited proliferation benefits as well. These findings provide an invaluable asset for stem cell-based tissue regeneration.
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Affiliation(s)
- Yiming Wang
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; Department of Joint Surgery, Shanghai East Hospital, Tongji University, Shanghai, China
| | - Yixuan Amy Pei
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Yuan Sun
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA
| | - Sheng Zhou
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA
| | - Xiao-Bing Zhang
- State Key Laboratory of Experimental Hematology, Tianjin, China; Department of Medicine, Loma Linda University, Loma Linda, CA, USA.
| | - Ming Pei
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA.
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23
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Piñeiro-Ramil M, Sanjurjo-Rodríguez C, Rodríguez-Fernández S, Castro-Viñuelas R, Hermida-Gómez T, Blanco-García FJ, Fuentes-Boquete I, Díaz-Prado S. Generation of Mesenchymal Cell Lines Derived from Aged Donors. Int J Mol Sci 2021; 22:10667. [PMID: 34639008 PMCID: PMC8508916 DOI: 10.3390/ijms221910667] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Revised: 09/21/2021] [Accepted: 09/29/2021] [Indexed: 02/07/2023] Open
Abstract
Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.
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Affiliation(s)
- María Piñeiro-Ramil
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
| | - Clara Sanjurjo-Rodríguez
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
- Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
| | - Silvia Rodríguez-Fernández
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
| | - Rocío Castro-Viñuelas
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
| | - Tamara Hermida-Gómez
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
- Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
- Grupo de Investigación en Reumatología (GIR), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario da Coruña (UDC-CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain
| | - Francisco J. Blanco-García
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
- Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
- Grupo de Investigación en Reumatología (GIR), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario da Coruña (UDC-CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain
| | - Isaac Fuentes-Boquete
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
- Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
| | - Silvia Díaz-Prado
- Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Servizo Galego de Saúde (SERGAS), 15006 A Coruña, Spain; (M.P.-R.); (C.S.-R.); (S.R.-F.); (R.C.-V.); (I.F.-B.)
- Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15071 A Coruña, Spain; (T.H.-G.); (F.J.B.-G.)
- Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
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Tan TT, Lai RC, Padmanabhan J, Sim WK, Choo ABH, Lim SK. Assessment of Tumorigenic Potential in Mesenchymal-Stem/Stromal-Cell-Derived Small Extracellular Vesicles (MSC-sEV). Pharmaceuticals (Basel) 2021; 14:ph14040345. [PMID: 33918628 PMCID: PMC8069985 DOI: 10.3390/ph14040345] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Revised: 03/29/2021] [Accepted: 04/07/2021] [Indexed: 12/11/2022] Open
Abstract
Mesenchymal-stem/stromal-cell-derived small extracellular vesicles (MSC-sEV) have been shown to ameliorate many diseases in preclinical studies. However, translating MSC-sEV into clinical use requires the development of scalable manufacturing processes for highly reproducible preparations of safe and potent MSC-sEVs. A major source of variability in MSC-sEV preparations is EV producer cells. To circumvent variability in producer cells, clonal immortalized MSC lines as EV producer lines are increasingly being used for sEV production. The use of sEVs from immortalized producer cells inevitably raises safety concerns regarding the tumorigenicity or tumor promoting potential of the EV products. In this study, cells from E1-MYC line, a MSC cell line immortalized with the MYC gene, were injected subcutaneously into athymic nude mice. At 84 days post-injection, no tumor formation was observed at the injection site, lungs, or lymph nodes. E1-MYC cells pre-and post-sEV production did not exhibit anchorage-independent growth in soft agar. Daily intraperitoneal injections of 1 or 5 μg sEVs from E1-MYC into athymic nude mice with FaDu human head and neck cancer xenografts for 28 days did not promote or inhibit tumor growth relative to the xenograft treated with vehicle control. Therefore, MYC-immortalized MSCs are not tumorigenic and sEVs from these MSCs do not promote tumor growth.
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Affiliation(s)
- Thong Teck Tan
- Institute of Molecular and Cellular Biology, A*STAR, 8A Biomedical Grove, Singapore 138648, Singapore; (T.T.T.); (R.C.L.); (W.K.S.)
| | - Ruenn Chai Lai
- Institute of Molecular and Cellular Biology, A*STAR, 8A Biomedical Grove, Singapore 138648, Singapore; (T.T.T.); (R.C.L.); (W.K.S.)
| | - Jayanthi Padmanabhan
- Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, Singapore 138668, Singapore; (J.P.); (A.B.H.C.)
| | - Wei Kian Sim
- Institute of Molecular and Cellular Biology, A*STAR, 8A Biomedical Grove, Singapore 138648, Singapore; (T.T.T.); (R.C.L.); (W.K.S.)
| | - Andre Boon Hwa Choo
- Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, Singapore 138668, Singapore; (J.P.); (A.B.H.C.)
- Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore (NUS), 9 Engineering Drive 1, Singapore 117575, Singapore
| | - Sai Kiang Lim
- Institute of Molecular and Cellular Biology, A*STAR, 8A Biomedical Grove, Singapore 138648, Singapore; (T.T.T.); (R.C.L.); (W.K.S.)
- Department of Surgery, YLL School of Medicine, National University of Singapore (NUS), 5 Lower Kent Ridge Road, Singapore 119074, Singapore
- Correspondence: ; Tel.: +65-64-070161
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25
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Zhu GQ, Jeon SH, Lee KW, Cho HJ, Ha US, Hong SH, Lee JY, Kwon EB, Kim HJ, Lee SM, Kim HY, Kim SW, Bae WJ. Engineered Stem Cells Improve Neurogenic Bladder by Overexpressing SDF-1 in a Pelvic Nerve Injury Rat Model. Cell Transplant 2021; 29:963689720902466. [PMID: 32067480 PMCID: PMC7444235 DOI: 10.1177/0963689720902466] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
There is still a lack of sufficient research on the mechanism behind neurogenic
bladder (NB) treatment. The aim of this study was to explore the effect of
overexpressed stromal cell-derived factor-1 (SDF-1) secreted by engineered
immortalized mesenchymal stem cells (imMSCs) on the NB. In this study, primary
bone marrow mesenchymal stem cells (BM-MSCs) were transfected into immortalized
upregulated SDF-1-engineered BM-MSCs (imMSCs/eSDF-1+) or immortalized normal SDF-1-engineered BM-MSCs
(imMSCs/eSDF-1−). NB rats induced by bilateral pelvic nerve (PN)
transection were treated with imMSCs/eSDF-1+, imMSCs/eSDF-1−, or sham. After a 4-week treatment, the bladder function was assessed by
cystometry and voiding pattern analysis. The PN and bladder tissues were
evaluated via immunostaining and western blotting analysis. We found that imMSCs/eSDF-1+ expressed higher levels of SDF-1 in vitro and in vivo. The treatment of imMSCs/eSDF-1+ improved NB and evidently stimulated the recovery of bladder wall in NB
rats. The recovery of injured nerve was more effective in the NB+imMSCs/eSDF-1+ group than in other groups. High SDF-1 expression improved the levels of
vascular endothelial growth factor and basic fibroblast growth factor. Apoptosis
was decreased after imMSCs injection, and was detected rarely in the NB+imMSCs/eSDF-1+ group. Injection of imMSCs boosted the expression of neuronal nitric
oxide synthase, p-AKT, and p-ERK in the NB+imMSCs/eSDF-1+ group than in other groups. Our findings demonstrated that overexpression
of SDF-1 induced additional MSC homing to the injured tissue, which improved the
NB by accelerating the restoration of injured nerve in a rat model.
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Affiliation(s)
- Guan Qun Zhu
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, The Catholic University of Korea, Seoul, Republic of Korea
| | - Seung Hwan Jeon
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, The Catholic University of Korea, Seoul, Republic of Korea
| | - Kyu Won Lee
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Hyuk Jin Cho
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - U-Syn Ha
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Sung-Hoo Hong
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Ji Youl Lee
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Eun Bi Kwon
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, The Catholic University of Korea, Seoul, Republic of Korea
| | - Hyo-Jin Kim
- Department of Stem Cell Therapy, SL BIGEN, Seongnam, Republic of Korea
| | - Soon Min Lee
- Department of Stem Cell Therapy, SL BIGEN, Seongnam, Republic of Korea
| | - Hey-Yon Kim
- Department of Stem Cell Therapy, SL BIGEN, Seongnam, Republic of Korea
| | - Sea Woong Kim
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, The Catholic University of Korea, Seoul, Republic of Korea
| | - Woong Jin Bae
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, The Catholic University of Korea, Seoul, Republic of Korea
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26
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Tian WJ, Jeon SH, Zhu GQ, Kwon EB, Kim GE, Bae WJ, Cho HJ, Ha US, Hong SH, Lee JY, Kim KS, Kim SW. Effect of high-BDNF microenvironment stem cells therapy on neurogenic bladder model in rats. Transl Androl Urol 2021; 10:345-355. [PMID: 33532323 PMCID: PMC7844501 DOI: 10.21037/tau-20-1072] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Background The purpose of this study is to explore the effects of high-BDNF microenvironment produced by engineered immortalized mesenchymal stem cells (imMSCs) on the neurogenic bladder (NB) and investigate underlying mechanism. Methods Male Sprague-Dawley rat (12-week-old, weighing about 370-400 g) were purchased from a Korean company (Orient Bio Co. Seongnam, Korea) and divided into the following groups (n=32): sham control group (n=8), NB group (n=8), NB + ImMSCs group (n=8), NB + ImMSCs (BDNF) group (n=8). The major pelvic ganglion (MPG) was observed under anesthesia. Three NB groups of rats were then subjected to bilateral MPG injury. The sham control group of rats was treated with sham surgery. Cystometry were performed before the rats were sacrificed, and then MPG and bladder were collected for histochemical and Western blot analysis. Results MSCs treatment improves lower urinary tract function, and the NB + ImMSCs (BDNF) group is better than the NB + ImMSCs group (P<0.01). MSCs treatment accelerates recovery of injured nerve tissue, and the NB + ImMSCs (BDNF) group is better than the NB + ImMSCs group (P<0.01). In high BDNF environment, apoptosis was reduced more significantly and muscle tissue recovered more rapidly (P<0.01). High-BDNF microenvironment activates more BDNF/TrkB/CREB signaling pathways (P<0.01). Conclusions In a rat NB model caused by nerve injury, imMSCs have certain effects on nerve tissue repair. At the same time, it was proved that increasing the expression of BDNF which had specific effect on nerve injury repair could more effectively repair injured MPG in local microenvironment. The mechanism may be related to the activation of the BDNF/TrkB/CREB signaling pathway and the reduction of apoptosis by highly expressed BDNF.
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Affiliation(s)
- Wen Jie Tian
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
| | - Seung Hwan Jeon
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
| | - Guan Qun Zhu
- Department of Urology, Affiliated Hospital of Qingdao University, Qingdao, China
| | - Eun Bi Kwon
- Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
| | - Ga Eun Kim
- Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
| | - Woong Jin Bae
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
| | - Hyuk Jin Cho
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - U-Syn Ha
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Sung-Hoo Hong
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Ji Youl Lee
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Kang Sup Kim
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Sae Woong Kim
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.,Catholic Integrative Medicine Research Institute, the Catholic University of Korea, Seoul, Republic of Korea
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27
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Jeon SH, Park MY. Transplantation of Brain-Derived Neurotrophic Factor-Expressing Mesenchymal Stem Cells Improves Lower Urinary Tract Symptoms in a Rat Model. KOREAN JOURNAL OF CLINICAL LABORATORY SCIENCE 2020. [DOI: 10.15324/kjcls.2020.52.4.417] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Affiliation(s)
- Seung Hwan Jeon
- Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Mi-Young Park
- Department of Clinical Laboratory Science, Suwon Science College, Hwaseong, Korea
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28
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Immortalizing Mesenchymal Stromal Cells from Aged Donors While Keeping Their Essential Features. Stem Cells Int 2020; 2020:5726947. [PMID: 32612662 PMCID: PMC7315279 DOI: 10.1155/2020/5726947] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Revised: 01/31/2020] [Accepted: 05/11/2020] [Indexed: 12/22/2022] Open
Abstract
Human bone marrow-derived mesenchymal stromal cells (MSCs) obtained from aged patients are prone to senesce and diminish their differentiation potential, therefore limiting their usefulness for osteochondral regenerative medicine approaches or to study age-related diseases, such as osteoarthiritis (OA). MSCs can be transduced with immortalizing genes to overcome this limitation, but transduction of primary slow-dividing cells has proven to be challenging. Methods for enhancing transduction efficiency (such as spinoculation, chemical adjuvants, or transgene expression inductors) can be used, but several parameters must be adapted for each transduction system. In order to develop a transduction method suitable for the immortalization of MSCs from aged donors, we used a spinoculation method. Incubation parameters of packaging cells, speed and time of centrifugation, and valproic acid concentration to induce transgene expression have been adjusted. In this way, four immortalized MSC lines (iMSC#6, iMSC#8, iMSC#9, and iMSC#10) were generated. These immortalized MSCs (iMSCs) were capable of bypassing senescence and proliferating at a higher rate than primary MSCs. Characterization of iMSCs showed that these cells kept the expression of mesenchymal surface markers and were able to differentiate towards osteoblasts, adipocytes, and chondrocytes. Nevertheless, alterations in the CD105 expression and a switch of cell fate-commitment towards the osteogenic lineage have been noticed. In conclusion, the developed transduction method is suitable for the immortalization of MSCs derived from aged donors. The generated iMSC lines maintain essential mesenchymal features and are expected to be useful tools for the bone and cartilage regenerative medicine research.
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29
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Comeau PA, Willett TL. Triethyleneglycol dimethacrylate addition improves the 3D-printability and construct properties of a GelMA-nHA composite system towards tissue engineering applications. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 112:110937. [PMID: 32409083 DOI: 10.1016/j.msec.2020.110937] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/09/2020] [Revised: 03/30/2020] [Accepted: 04/05/2020] [Indexed: 12/26/2022]
Abstract
In tissue engineering, there is a growing interest in the development of 3D printable bone tissue-inspired nanocomposites. However, most such nanocomposites have poor mechanical properties, owing to poor dispersion of the mineral phase (e.g. nano-hydroxyapatite, nHA) within the organic phase (e.g. methacrylated gelatin, GelMA) and low volume fractions of each phase. Triethyleneglycol dimethacrylate (TEGDMA) is commonly added to dental resin-based composites to improve the properties of the dental resin. Here, the effects of substituting a portion of the water phase in a GelMA-nHA composite with TEGDMA were evaluated. TEGDMA improved the dispersion of nHA within the highly-concentrated GelMA-based composite ink, as well as increased the ink's shear yield strength and reduced the critical energy for ink cure. As a result, the printability of the composite ink was greatly improved upon TEGDMA inclusion. Lastly, while the swelling of the cast composite in 37 °C water increased slightly, the mechanical properties (tensile strength, toughness, and stiffness) of the cast composite increased by at least an order of magnitude upon TEGDMA addition, and all composites demonstrated MSC cytocompatibility after 24 h. Overall, TEGDMA shows promise as an additive to tune properties of the GelMA-nHA system towards use in tissue engineering applications.
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Affiliation(s)
- P A Comeau
- 200 University Avenue West, Systems Design Engineering, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada
| | - T L Willett
- 200 University Avenue West, Systems Design Engineering, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada.
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30
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Seok J, Jung HS, Park S, Lee JO, Kim CJ, Kim GJ. Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells. Stem Cell Res Ther 2020; 11:1. [PMID: 31900237 PMCID: PMC6941254 DOI: 10.1186/s13287-019-1471-y] [Citation(s) in RCA: 231] [Impact Index Per Article: 46.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 10/10/2019] [Accepted: 10/25/2019] [Indexed: 02/08/2023] Open
Abstract
Background Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations. Methods Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation. Results Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-β-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs. Conclusions These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.
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Affiliation(s)
- Jin Seok
- Department of Biomedical Science, CHA University, 689, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea
| | - Hyun Sook Jung
- Department of Biomedical Science, CHA University, 689, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea
| | - Sohae Park
- Department of Biomedical Science, CHA University, 689, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea
| | - Jung Ok Lee
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
| | - Chong Jai Kim
- Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
| | - Gi Jin Kim
- Department of Biomedical Science, CHA University, 689, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
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Rock Inhibitor Y-27632 Enables Feeder-Free, Unlimited Expansion of Sus scrofa domesticus Swine Airway Stem Cells to Facilitate Respiratory Research. Stem Cells Int 2019; 2019:3010656. [PMID: 31871466 PMCID: PMC6906834 DOI: 10.1155/2019/3010656] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Accepted: 10/29/2019] [Indexed: 12/21/2022] Open
Abstract
Current limitations in the efficacy of treatments for chronic respiratory disorders position them as prospective regenerative medicine therapeutic targets. A substantial barrier to these ambitions is that research requires large numbers of cells whose acquisition is hindered by the limited availability of human tissue samples leading to an overreliance on physiologically dissimilar rodents. The development of cell culture strategies for airway cells from large mammals will more effectively support the transition from basic research to clinical therapy. Using readily available porcine lungs, we isolated conducting airway tissue and subsequently a large number of porcine airway epithelial cells (pAECs) using a digestion and mechanical scraping technique. Cells were cultured in a variety of culture media formulations, both foetal bovine serum-containing and serum-free media, in air (21%) and physiological (2%) oxygen tension and in the presence and absence of Rho kinase inhibitor Y-27362 (RI). Cell number at isolation and subsequent population doublings were determined; cells were characterised during culture and following differentiation by immunofluorescence, histology, and IL-8 ELISA. Cells were positive for epithelial markers (pan-cytokeratin and E-cadherin) and negative for fibroblastic markers (vimentin and smooth muscle actin). Supplementation of cultures with Y-27632 allowed for unlimited expansion whilst sustaining an epithelial phenotype. Early passage pAECs readily produced differentiated air-liquid interface (ALI) cultures with a capacity for mucociliary differentiation retained after substantial expansion, strongly modulated by the culture condition applied. Primary pAECs will be a useful tool to further respiratory-oriented research whilst RI-expanded pAECs are a promising tool, particularly with further optimisation of culture conditions.
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Facchin F, Alviano F, Canaider S, Bianconi E, Rossi M, Bonsi L, Casadei R, Biava PM, Ventura C. Early Developmental Zebrafish Embryo Extract to Modulate Senescence in Multisource Human Mesenchymal Stem Cells. Int J Mol Sci 2019; 20:2646. [PMID: 31146388 PMCID: PMC6600478 DOI: 10.3390/ijms20112646] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/24/2019] [Accepted: 05/25/2019] [Indexed: 12/14/2022] Open
Abstract
Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated β-galactosidase (SA β-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.
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Affiliation(s)
- Federica Facchin
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
- National Laboratory of Molecular Biology and Stem Cell Bioengineering of the National Institute of Biostructures and Biosystems (NIBB)-Eldor Lab, at the Innovation Accelerator, CNR, Via Piero Gobetti 101, 40129 Bologna, Italy.
| | - Francesco Alviano
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
| | - Silvia Canaider
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
- National Laboratory of Molecular Biology and Stem Cell Bioengineering of the National Institute of Biostructures and Biosystems (NIBB)-Eldor Lab, at the Innovation Accelerator, CNR, Via Piero Gobetti 101, 40129 Bologna, Italy.
| | - Eva Bianconi
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
- National Laboratory of Molecular Biology and Stem Cell Bioengineering of the National Institute of Biostructures and Biosystems (NIBB)-Eldor Lab, at the Innovation Accelerator, CNR, Via Piero Gobetti 101, 40129 Bologna, Italy.
| | - Martina Rossi
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
| | - Laura Bonsi
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
| | - Raffaella Casadei
- Department for Life Quality Studies (QuVi), University of Bologna, Corso D'Augusto 237, 47921 Rimini, Italy.
| | - Pier Mario Biava
- Scientific Institute of Research and Care Multimedica, Via Milanese 300, 20099 Sesto San Giovanni (Milano), Italy.
| | - Carlo Ventura
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
- National Laboratory of Molecular Biology and Stem Cell Bioengineering of the National Institute of Biostructures and Biosystems (NIBB)-Eldor Lab, at the Innovation Accelerator, CNR, Via Piero Gobetti 101, 40129 Bologna, Italy.
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Kazlauskas A, Darinskas A, Meškys R, Tamašauskas A, Urbonavičius J. Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy. BMC Cancer 2019; 19:197. [PMID: 30832616 PMCID: PMC6399854 DOI: 10.1186/s12885-019-5409-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Accepted: 02/26/2019] [Indexed: 01/11/2023] Open
Abstract
Background The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. Methods Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. Results 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 μM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. Conclusions We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy. Electronic supplementary material The online version of this article (10.1186/s12885-019-5409-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Arunas Kazlauskas
- Laboratory of Molecular Neurooncology, Neuroscience Institute, Medical Academy, Lithuanian University of Health Sciences, Eiveniu str. 4, LT-50161, Kaunas, Lithuania.
| | - Adas Darinskas
- Laboratory of Immunology, National Cancer Institute, Santariskiu Str. 1, LT-08660, Vilnius, Lithuania
| | - Rolandas Meškys
- Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al.7, LT-10222, Vilnius, Lithuania
| | - Arimantas Tamašauskas
- Laboratory of Molecular Neurooncology, Neuroscience Institute, Medical Academy, Lithuanian University of Health Sciences, Eiveniu str. 4, LT-50161, Kaunas, Lithuania
| | - Jaunius Urbonavičius
- Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al.7, LT-10222, Vilnius, Lithuania.,Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Sauletekio al.11, LT-10221, Vilnius, Lithuania
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Burk J, Holland H, Lauermann AF, May T, Siedlaczek P, Charwat V, Kasper C. Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line. Biotechnol Bioeng 2019; 116:1417-1426. [PMID: 30739319 DOI: 10.1002/bit.26950] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2018] [Revised: 02/01/2019] [Accepted: 02/06/2019] [Indexed: 12/12/2022]
Abstract
Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.
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Affiliation(s)
- Janina Burk
- Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Heidrun Holland
- Saxon Incubator for Clinical Translation (SIKT), University of Leipzig, Leipzig, Germany
| | - Anne F Lauermann
- Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
| | | | - Philipp Siedlaczek
- Institute of Physics and Materials Science, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Verena Charwat
- Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Cornelia Kasper
- Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
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Abstract
Objective: Gliomas are the most common neoplasm of the central nervous system (CNS); however, traditional imaging techniques do not show the boundaries of tumors well. Some researchers have found a new therapeutic mode to combine nanoparticles, which are nanosized particles with various properties for specific therapeutic purposes, and stem cells for tracing gliomas. This review provides an introduction of the basic understanding and clinical applications of the combination of stem cells and nanoparticles as a contrast agent for glioma imaging. Data Sources: Studies published in English up to and including 2017 were extracted from the PubMed database with the selected key words of “stem cell,” “glioma,” “nanoparticles,” “MRI,” “nuclear imaging,” and “Fluorescence imaging.” Study Selection: The selection of studies focused on both preclinical studies and basic studies of tracking glioma with nanoparticle-labeled stem cells. Results: Studies have demonstrated successful labeling of stem cells with multiple types of nanoparticles. These labeled stem cells efficiently migrated to gliomas of varies models and produced signals sensitively captured by different imaging modalities. Conclusion: The use of nanoparticle-labeled stem cells is a promising imaging platform for the tracking and treatment of gliomas.
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Affiliation(s)
- Shuang-Lin Deng
- Department of Neurosurgical Oncology, The First Hospital of Jilin University, Changchun, Jilin 130021, China
| | - Yun-Qian Li
- Department of Neurosurgical Oncology, The First Hospital of Jilin University, Changchun, Jilin 130021, China
| | - Gang Zhao
- Department of Neurosurgical Oncology, The First Hospital of Jilin University, Changchun, Jilin 130021, China
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Twine NA, Harkness L, Adjaye J, Aldahmash A, Wilkins MR, Kassem M. Molecular Phenotyping of Telomerized Human Bone Marrow Skeletal Stem Cells Reveals a Genetic Program of Enhanced Proliferation and Maintenance of Differentiation Responses. JBMR Plus 2018; 2:257-267. [PMID: 30283907 PMCID: PMC6139702 DOI: 10.1002/jbm4.10050] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 03/15/2018] [Accepted: 03/24/2018] [Indexed: 12/15/2022] Open
Abstract
Long-term in vitro expansion of bone marrow stromal (skeletal) stem cells (also known as human mesenchymal stem cells [hMSC]) is associated with replicative senescence and impaired functions. We have previously reported that telomerization of hMSC through hTERT overexpression led to bypassing a replicative senescence phenotype and improved in vitro and in vivo functions. However, the molecular consequence of telomerization is poorly characterized. Thus, we compared the molecular phenotype of a well-studied telomerized hMSC (hMSC-TERT) cell line with primary hMSC. At a cellular level, both cell populations exhibited strong concordance for the known hMSC CD markers, similar responses to osteoblast (OB) differentiation induction, and formed heterotopic bone in vivo. Overall gene expression was highly correlated between both cell types with an average Pearson's correlation coefficient (R2) between the gene expression of all primary hMSC and all hMSC-TERT samples of 0.95 (range 0.93-0.96). Quantitative analysis of gene expression of CD markers, OB cell markers, and transcription factors (TF) showed a high degree of similarity between the two cell populations (72%, 77%, and 81%, respectively). The hMSC-TERT population was enriched mainly for genes associated with cell cycle and cell cycle signaling when compared with primary hMSC. Other enrichment was observed for genes involved in cell adhesion and skeletal system development and immune response pathways. Interestingly, hMSC-TERT shared a telomerization signature with upregulation of cancer/testis antigens, MAGE, and PAGE genes. Our data demonstrate that the enhanced biological characteristics of hMSC after telomerization are mainly due to enhanced expression of cell proliferation genes, whereas gene expression responses to differentiation are maintained. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
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Affiliation(s)
- Natalie A Twine
- Systems Biology InitiativeSchool of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
- CSIROSydneyAustralia
| | - Linda Harkness
- Department of Endocrinology and MetabolismEndocrine Research Laboratory (KMEB)Odense University HospitalOdenseDenmark
| | - James Adjaye
- Institute for Stem Cell Research and Regenerative MedicineFaculty of MedicineHeinrich Heine UniversityDüsseldorfGermany
| | - Abdullah Aldahmash
- Stem Cell UnitDepartment of Anatomy, Faculty of MedicineKing Saud UniversityRiyadhSaudi Arabia
| | - Marc R Wilkins
- Systems Biology InitiativeSchool of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
| | - Moustapha Kassem
- Systems Biology InitiativeSchool of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
- Department of Endocrinology and MetabolismEndocrine Research Laboratory (KMEB)Odense University HospitalOdenseDenmark
- Stem Cell UnitDepartment of Anatomy, Faculty of MedicineKing Saud UniversityRiyadhSaudi Arabia
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How cell culture conditions affect the microstructure and nanomechanical properties of extracellular matrix formed by immortalized human mesenchymal stem cells: An experimental and modelling study. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2018; 89:149-159. [DOI: 10.1016/j.msec.2018.03.027] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Revised: 02/02/2018] [Accepted: 03/26/2018] [Indexed: 12/27/2022]
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Dale TP, Forsyth NR. Ectopic Telomerase Expression Fails to Maintain Chondrogenic Capacity in Three-Dimensional Cultures of Clinically Relevant Cell Types. Biores Open Access 2018; 7:10-24. [PMID: 29588876 PMCID: PMC5865620 DOI: 10.1089/biores.2018.0008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The poor healing capacity of cartilage and lack of effective treatment for associated disease and trauma makes it a strong candidate for a regenerative medicine approach. Potential therapies tested to date, although effective, have met with a number of intrinsic difficulties possibly related to limited autologous chondrocyte cell yield and quality of cartilage produced. A potential mechanism to bypass limited cell yields and improve quality of differentiation is to immortalize relevant cell types through the ectopic expression of telomerase. Pellet cultures of human chondrocytes (OK3), bone marrow mesenchymal stem cells (BMA13), and embryonic stem cell (H1 line)-derived cells (1C6) and their human telomerase reverse transcriptase (hTERT) transduced counterparts were maintained for 20 days in standard maintenance medium (MM) or transforming growth factor-β3-supplemented prochondrogenic medium (PChM). Pellets were assessed for volume and density by microcomputed tomography. Quantitative gene expression (COL1A2, COL2A1, COL3A1, COL6A3, COL10A1, ACAN, COMP, SOX9); sulfated glycosaminoglycans (sGAGs), and DNA quantification were performed. Histology and immunohistochemistry were used to determine matrix constituent distribution. Pellet culture in PChM resulted in significantly larger pellets with an overall increased density when compared with MM culture. Gene expression analysis revealed similarities in expression patterns between telomerase-transduced and parental cells in both MM and PChM. Of the three parental cell types examined OK3 and BMA13 produced similar amounts of pellet-associated sGAG in PChM (4.62 ± 1.20 and 4.91 ± 1.37 μg, respectively) with lower amounts in 1C6 (2.89 ± 0.52 μg), corresponding to 3.1, 2.3, and 1.6-fold increases from day 0. In comparison, telomerase-transduced cells all had much lower sGAG with OK3H at 2.74 ± 0.11 μg, BMA13H 1.29 ± 0.34 μg, and 1C6H 0.52 ± 0.01 μg corresponding to 1.2, 0.87, and 0.34-fold changes compared with day 0. Histology of day 20 pellets displayed reduced staining overall for collagens and sGAG in telomerase-transduced cells, most notably with alterations in aggrecan and collagen VI; all cells stained positively for collagen II. We conclude that while telomerase transduction may be an effective technique to extend cellular proliferative capacity, it is not sufficient in isolation to sustain a naive chondrogenic phenotype across multiple cell types.
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Affiliation(s)
- Tina P Dale
- Faculty of Medicine and Health Sciences, Guy Hilton Research Center, Institute for Science and Technology in Medicine, Keele University, Staffordshire, United Kingdom
| | - Nicholas R Forsyth
- Faculty of Medicine and Health Sciences, Guy Hilton Research Center, Institute for Science and Technology in Medicine, Keele University, Staffordshire, United Kingdom
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Mesenchymal Stem Cells: Cell Fate Decision to Osteoblast or Adipocyte and Application in Osteoporosis Treatment. Int J Mol Sci 2018; 19:ijms19020360. [PMID: 29370110 PMCID: PMC5855582 DOI: 10.3390/ijms19020360] [Citation(s) in RCA: 293] [Impact Index Per Article: 41.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Revised: 01/13/2018] [Accepted: 01/22/2018] [Indexed: 12/11/2022] Open
Abstract
Osteoporosis is a progressive skeletal disease characterized by decreased bone mass and degraded bone microstructure, which leads to increased bone fragility and risks of bone fracture. Osteoporosis is generally age related and has become a major disease of the world. Uncovering the molecular mechanisms underlying osteoporosis and developing effective prevention and therapy methods has great significance for human health. Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into osteoblasts, adipocytes, or chondrocytes, and have become the favorite source of cell-based therapy. Evidence shows that during osteoporosis, a shift of the cell differentiation of MSCs to adipocytes rather than osteoblasts partly contributes to osteoporosis. Thus, uncovering the molecular mechanisms of the osteoblast or adipocyte differentiation of MSCs will provide more understanding of MSCs and perhaps new methods of osteoporosis treatment. The MSCs have been applied to both preclinical and clinical studies in osteoporosis treatment. Here, we review the recent advances in understanding the molecular mechanisms regulating osteoblast differentiation and adipocyte differentiation of MSCs and highlight the therapeutic application studies of MSCs in osteoporosis treatment. This will provide researchers with new insights into the development and treatment of osteoporosis.
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Teng SW, Lo YS, Liu WT, Hsuan Y, Lin W. A genome-wide comparison of mesenchymal stem cells derived from human placenta and umbilical cord. Taiwan J Obstet Gynecol 2017; 56:664-671. [DOI: 10.1016/j.tjog.2017.08.016] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/13/2017] [Indexed: 12/29/2022] Open
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Husak Z, Dworzak MN. Chronic stress induces CD99, suppresses autophagy, and affects spontaneous adipogenesis in human bone marrow stromal cells. Stem Cell Res Ther 2017; 8:83. [PMID: 28420430 PMCID: PMC5395812 DOI: 10.1186/s13287-017-0532-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Revised: 02/20/2017] [Accepted: 03/09/2017] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Bone marrow-derived mesenchymal stromal cells (MSCs) are multipotent cells with a high constitutive level of autophagy and low expression of CD99. Under certain conditions, MSCs may develop tumorigenic properties. However, these transformation-induced conditions are largely unknown. Recently, we have identified an association between Hsp70, a main participant in cellular stress response and tumorigenesis, and CD99. Preliminary observations had revealed upregulation of both proteins in stressed long-term cultured MSCs. And so we hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation in MSCs. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in their survival. METHODS Human telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone marrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on growth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry, differentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic examination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting, and reverse transcriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology examination and western blotting. RESULTS Chronic stress factors are able to change the morphology of MSCs and to inhibit spontaneous differentiation into adipocyte lineage. Furthermore, CD99 elevation and downregulation of p53 and p21 accompanied defective autophagy, which is usually associated with tumor formation. We found that inhibition of autophagy by chloroquine promoted cell detachment and modulated CD99 expression level whereas incorporation of CD99 recombinant protein into the cells suppressed autophagy. CONCLUSIONS Obtained results provide a model for chronic stress-induced transformation of MSCs via CD99 and may therefore be highly relevant to mesenchymal tumorigenesis.
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Affiliation(s)
- Zvenyslava Husak
- St. Anna Kinderkrebsforschung, Children’s Cancer Research Institute, Zimmermannplatz 10, 1090 Vienna, Austria
| | - Michael N. Dworzak
- St. Anna Kinderkrebsforschung, Children’s Cancer Research Institute, Zimmermannplatz 10, 1090 Vienna, Austria
- St. Anna Kinderspital, Kinderspitalgasse 6, 1090 Vienna, Austria
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Tao K, Rey-Rico A, Frisch J, Venkatesan JK, Schmitt G, Madry H, Lin J, Cucchiarini M. Effects of combined rAAV-mediated TGF-β and sox9 gene transfer and overexpression on the metabolic and chondrogenic activities in human bone marrow aspirates. J Exp Orthop 2017; 4:4. [PMID: 28176272 PMCID: PMC5296264 DOI: 10.1186/s40634-017-0077-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/21/2016] [Accepted: 01/16/2017] [Indexed: 02/08/2023] Open
Abstract
Background Transplantation of genetically modified bone marrow concentrates is an attractive approach to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage. Methods Human bone marrow aspirates were co-transduced with recombinant adeno-associated virus (rAAV) vectors to overexpress the pleiotropic transformation growth factor beta (TGF-β) and the cartilage-specific transcription factor sox9 as a means to enhance the chondroreparative processes in conditions of specific lineage differentiation. Results Successful TGF-β/sox9 combined gene transfer and overexpression via rAAV was achieved in chondrogenically induced human bone marrow aspirates for up to 21 days, the longest time point evaluated, leading to increased proliferation, matrix synthesis, and chondrogenic differentiation relative to control treatments (reporter lacZ treatment, absence of vector application) especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation in the aspirates. Conclusions These findings report the possibility of directly modifying bone marrow aspirates by combined therapeutic gene transfer as a potent and convenient future approach to improve the repair of articular cartilage lesions.
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Affiliation(s)
- Ke Tao
- Institute of Arthritis, Peking University People's Hospital, No. 11 Xizhimen Nan Road, Xicheng District, Beijing, 100044, People's Republic of China.,Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany
| | - Ana Rey-Rico
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany
| | - Janina Frisch
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany
| | - Jagadeesh Kumar Venkatesan
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany
| | - Gertrud Schmitt
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany
| | - Henning Madry
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany.,Department of Orthopaedic Surgery, Saarland University Medical Center, Homburg/Saar, Germany
| | - Jianhao Lin
- Institute of Arthritis, Peking University People's Hospital, No. 11 Xizhimen Nan Road, Xicheng District, Beijing, 100044, People's Republic of China.
| | - Magali Cucchiarini
- Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany.
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Frisch J, Cucchiarini M. Gene- and Stem Cell-Based Approaches to Regulate Hypertrophic Differentiation in Articular Cartilage Disorders. Stem Cells Dev 2016; 25:1495-1512. [DOI: 10.1089/scd.2016.0106] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Affiliation(s)
- Janina Frisch
- Center of Experimental Orthopaedics, Saarland University and Saarland University Medical Center, Homburg, Germany
| | - Magali Cucchiarini
- Center of Experimental Orthopaedics, Saarland University and Saarland University Medical Center, Homburg, Germany
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Phetfong J, Sanvoranart T, Nartprayut K, Nimsanor N, Seenprachawong K, Prachayasittikul V, Supokawej A. Osteoporosis: the current status of mesenchymal stem cell-based therapy. Cell Mol Biol Lett 2016; 21:12. [PMID: 28536615 PMCID: PMC5414670 DOI: 10.1186/s11658-016-0013-1] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Accepted: 01/25/2016] [Indexed: 12/21/2022] Open
Abstract
Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.
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Affiliation(s)
- Jitrada Phetfong
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Tanwarat Sanvoranart
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Kuneerat Nartprayut
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Natakarn Nimsanor
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Kanokwan Seenprachawong
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Virapong Prachayasittikul
- Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
| | - Aungkura Supokawej
- Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Phuttamonthon, Salaya, Nakhon Pathom 73170 Thailand
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45
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Amin ARMR, Karpowicz PA, Carey TE, Arbiser J, Nahta R, Chen ZG, Dong JT, Kucuk O, Khan GN, Huang GS, Mi S, Lee HY, Reichrath J, Honoki K, Georgakilas AG, Amedei A, Amin A, Helferich B, Boosani CS, Ciriolo MR, Chen S, Mohammed SI, Azmi AS, Keith WN, Bhakta D, Halicka D, Niccolai E, Fujii H, Aquilano K, Ashraf SS, Nowsheen S, Yang X, Bilsland A, Shin DM. Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds. Semin Cancer Biol 2015; 35 Suppl:S55-S77. [PMID: 25749195 PMCID: PMC4561219 DOI: 10.1016/j.semcancer.2015.02.005] [Citation(s) in RCA: 72] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2014] [Revised: 02/11/2015] [Accepted: 02/13/2015] [Indexed: 12/14/2022]
Abstract
The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.
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Affiliation(s)
| | - Phillip A Karpowicz
- Department of Biological Sciences, University of Windsor, 401 Sunset Ave., Room 327, Windsor, Ontario, N9B 3P4, Canada
| | | | - Jack Arbiser
- Winship Cancer Institute of Emory University, Atlanta, GA, USA; Atlanta Veterans Administration Health Center, Atlanta, GA, USA
| | - Rita Nahta
- Winship Cancer Institute of Emory University, Atlanta, GA, USA
| | - Zhuo G Chen
- Winship Cancer Institute of Emory University, Atlanta, GA, USA
| | - Jin-Tang Dong
- Winship Cancer Institute of Emory University, Atlanta, GA, USA
| | - Omer Kucuk
- Winship Cancer Institute of Emory University, Atlanta, GA, USA
| | | | | | - Shijun Mi
- Albert Einstein College of Medicine, New York, NY, USA
| | - Ho-Young Lee
- College of Pharmacy, Seoul National University, Seoul, South Korea
| | | | | | | | | | - Amr Amin
- UAE University, Al Ain, United Arab Emirates; Faculty of Science, Cairo University, Cairo, Egypt
| | - Bill Helferich
- University of Illinois at Urbana Champaign, Urbana Champaign, IL, USA
| | | | | | - Sophie Chen
- Ovarian and Prostate Cancer Research Laboratory, Guildford, Surrey, United Kingdom
| | | | | | | | - Dipita Bhakta
- School of Chemical and Bio Technology, SASTRA University, Thanjavur, India
| | | | | | | | | | | | - Somaira Nowsheen
- Medical Scientist Training Program, Mayo Medical School, Mayo Graduate School, Mayo Clinic, Rochester, MN, USA
| | - Xujuan Yang
- University of Illinois at Urbana Champaign, Urbana Champaign, IL, USA
| | | | - Dong M Shin
- Winship Cancer Institute of Emory University, Atlanta, GA, USA.
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46
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He S, Li Y, Chen Y, Zhu Y, Zhang X, Xia X, Sun H. Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase. Cytotechnology 2015; 68:1435-45. [PMID: 26341227 DOI: 10.1007/s10616-015-9903-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2014] [Accepted: 07/20/2015] [Indexed: 11/27/2022] Open
Abstract
Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.
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Affiliation(s)
- Shan He
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yangyang Li
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yang Chen
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yue Zhu
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Xinyu Zhang
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Xiaoli Xia
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Huaichang Sun
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China.
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47
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Frisch J, Rey-Rico A, Venkatesan JK, Schmitt G, Madry H, Cucchiarini M. Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates upon rAAV-Mediated Gene Transfer and Overexpression of the Insulin-Like Growth Factor I. Tissue Eng Part A 2015; 21:2460-71. [PMID: 26123891 DOI: 10.1089/ten.tea.2014.0679] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Direct therapeutic gene transfer in marrow concentrates is an attractive strategy to conveniently enhance the chondrogenic differentiation processes as a means to improve the healing response of damaged articular cartilage upon reimplantation in sites of injury. In the present study, we evaluated the ability of the clinically adapted recombinant adeno-associated virus (rAAV) vectors to mediate overexpression of the insulin-like growth factor I (IGF-I) in human bone marrow aspirates that may modulate the proliferative, anabolic activities, and chondrogenic differentiation potential in such samples in vitro. The results demonstrate that successful, significant rAAV-mediated IGF-I gene transfer and expression were achieved in transduced aspirates (up to 105.9±35.1 pg rhIGF-I/mg total proteins) over time (21 days) at very high levels (∼80% of cells expressing the candidate IGF-I transgene), leading to increased levels of proliferation, matrix synthesis, and chondrogenic differentiation over time compared with the control (lacZ) condition. Treatment with the candidate IGF-I vector also stimulated the hypertrophic and osteogenic differentiation processes in the aspirates, suggesting that the regulation of IGF-I expression through rAAV will be a prerequisite for future translation of the approach in vivo. However, these findings show the possible benefits of this vector class to directly modify marrow concentrates as a convenient tool for strategies that aim at improving the repair of articular cartilage lesions.
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Affiliation(s)
- Janina Frisch
- 1 Center of Experimental Orthopaedics, Saarland University Medical Center , Homburg/Saar, Germany
| | - Ana Rey-Rico
- 1 Center of Experimental Orthopaedics, Saarland University Medical Center , Homburg/Saar, Germany
| | | | - Gertrud Schmitt
- 1 Center of Experimental Orthopaedics, Saarland University Medical Center , Homburg/Saar, Germany
| | - Henning Madry
- 1 Center of Experimental Orthopaedics, Saarland University Medical Center , Homburg/Saar, Germany .,2 Department of Orthopedic Surgery, Saarland University Medical Center , Homburg/Saar, Germany
| | - Magali Cucchiarini
- 1 Center of Experimental Orthopaedics, Saarland University Medical Center , Homburg/Saar, Germany
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48
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Amiri F, Jahanian-Najafabadi A, Roudkenar MH. In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments : In vitro augmentation of mesenchymal stem cells viability. Cell Stress Chaperones 2015; 20:237-51. [PMID: 25527070 PMCID: PMC4326383 DOI: 10.1007/s12192-014-0560-1] [Citation(s) in RCA: 85] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2014] [Revised: 12/02/2014] [Accepted: 12/07/2014] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are under intensive investigation for use in cell-based therapies because their differentiation abilities, immunomodulatory effects, and homing properties offer potential for significantly augmenting regenerative capacity of many tissues. Nevertheless, major impediments to their therapeutic application, such as low proliferation and survival rates remain as obstacles to broad clinical use of MSCs. Another major challenge to evolution of MSC-based therapies is functional degradation of these cells as a result of their exposure to oxidative stressors during isolation. Indeed, oxidative stress-mediated MSC depletion occurs due to inflammatory processes associated with chemotherapy, radiotherapy, and expression of pro-apoptotic factors, and the microenvironment of damaged tissue in patients receiving MSC therapy is typically therapeutic not favorable to their survival. For this reason, any strategies that enhance the viability and proliferative capacity of MSCs associated with their therapeutic use are of great value. Here, recent strategies used by various researchers to improve MSC allograft function are reviewed, with particular focus on in vitro conditioning of MSCs in preparation for clinical application. Preconditioning, genetic manipulation, and optimization of MSC culture conditions are some examples of the methodologies described in the present article, along with novel strategies such as treatment of MSCs with secretome and MSC-derived microvesicles. This topic material is likely to find value as a guide for both research and clinical use of MSC allografts and for improvement of the value that use of these cells brings to health care.
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Affiliation(s)
- Fatemeh Amiri
- />Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
| | - Ali Jahanian-Najafabadi
- />Department of Pharmaceutical Biotechnology, School of Pharmacy, Isfahan University of Medical Sciences and Health Services, Isfahan, Iran
| | - Mehryar Habibi Roudkenar
- />Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
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49
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Skårn M, Noordhuis P, Wang MY, Veuger M, Kresse SH, Egeland EV, Micci F, Namløs HM, Håkelien AM, Olafsrud SM, Lorenz S, Haraldsen G, Kvalheim G, Meza-Zepeda LA, Myklebost O. Generation and characterization of an immortalized human mesenchymal stromal cell line. Stem Cells Dev 2014; 23:2377-89. [PMID: 24857590 PMCID: PMC4172386 DOI: 10.1089/scd.2013.0599] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Accepted: 05/14/2014] [Indexed: 12/31/2022] Open
Abstract
Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and high-resolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.
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Affiliation(s)
- Magne Skårn
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Paul Noordhuis
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Meng-Yu Wang
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Marjan Veuger
- Section of Vascular Endothelial Cells, Laboratory of Immunohistochemistry and Immunopathology, Rikshospitalet, Oslo University Hospital, Oslo, Norway
| | - Stine Henrichson Kresse
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Eivind Valen Egeland
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Francesca Micci
- Section for Cancer Cytogenetics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Heidi Maria Namløs
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Anne-Mari Håkelien
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Solveig Mjelstad Olafsrud
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Genomics Core Facility, Oslo University Hospital, Oslo, Norway
| | - Susanne Lorenz
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Genomics Core Facility, Oslo University Hospital, Oslo, Norway
| | - Guttorm Haraldsen
- Section of Vascular Endothelial Cells, Laboratory of Immunohistochemistry and Immunopathology, Rikshospitalet, Oslo University Hospital, Oslo, Norway
| | - Gunnar Kvalheim
- Department of Cell Therapy, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Leonardo Andrés Meza-Zepeda
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Genomics Core Facility, Oslo University Hospital, Oslo, Norway
| | - Ola Myklebost
- Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Genomics Core Facility, Oslo University Hospital, Oslo, Norway
- Norwegian Center for Stem Cell Research, Oslo University Hospital, Oslo, Norway
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50
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Use of Tissue Engineering Strategies to Repair Joint Tissues in Osteoarthritis: Viral Gene Transfer Approaches. Curr Rheumatol Rep 2014; 16:449. [DOI: 10.1007/s11926-014-0449-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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