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Silvano A, Sotillo J, Cecchi M, Loukas A, Ouedraogo M, Parenti A, Bruschi F, Torcia MG, Mangano VD. Schistosoma heamatobium tetraspanins TSP-2 and TSP-6 induce Dendritic Cells maturation, cytokine production and T helper cells differentiation in vitro. Microbes Infect 2025; 27:105439. [PMID: 39549890 DOI: 10.1016/j.micinf.2024.105439] [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] [Received: 06/27/2024] [Revised: 11/13/2024] [Accepted: 11/13/2024] [Indexed: 11/18/2024]
Abstract
Urogenital schistosomiasis caused by Schistosoma haematobium is a major cause of disability in endemic areas. Despite its socio-economic burden, no vaccine exists and the parasite's immunobiology remains underexplored. Genome annotation has revealed over 40 different genes encoding tetraspanins, transmembrane proteins with known immunomodulatory properties in other plathelminthes. This study investigated the role of Sh-TSP-2, Sh-TSP-6 and Sh-TSP-23, which are expressed in the parasite's tegument and extracellular vesicles (EVs). Immature dendritic cells (DCs) from unexposed healthy donors were stimulated with these proteins to evaluate maturation maker expression and cytokine production. Also, pre-activated T CD4+ cells were stimulated with the DCs supernatant to assess cytokine gene expression. Sh-TSP-2 and Sh-TSP-6 induced maturation markers and cytokine production in DCs: Sh-TSP-2 increased CD80 and CD83 levels and the concentration of both pro-inflammatory (IL-6, TNF) and regulatory (IL-10) cytokines, while Sh-TSP-6 increased the production of IL-6. Moreover, supernatants from Sh-TSP-2 stimulated DCs induced the expression of Th1 (IFNɣ) and regulatory (IL-10) cytokines in CD4+ T cells, while Sh-TSP-6 induced Th2 (IL-4, IL-13) cytokine expression. These results provide evidence that S. haematobium tetraspanins modulate the response of human DCs and CD4+ T cells in vitro, and support Sh-TSP-2 as a promising vaccine candidate.
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Affiliation(s)
- Angela Silvano
- Dept. of Health Sciences, University of Florence, Florence, Italy
| | - Javier Sotillo
- Parasitology Reference and Research Laboratory, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
| | - Marta Cecchi
- Dept. of Health Sciences, University of Florence, Florence, Italy; Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy
| | - Alex Loukas
- Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia
| | - Mireille Ouedraogo
- Dept. of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy; Dep. of Public Health and Infectious Diseases, University of Rome La Sapienza, Italy; Centre National de Recherche et Formation sur le Paludisme, Ouagadougou, Burkina Faso
| | - Astrid Parenti
- Dept. of Health Sciences, University of Florence, Florence, Italy
| | - Fabrizio Bruschi
- Dept. of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
| | | | - Valentina D Mangano
- Dept. of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy.
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Song M, Yang X, Zhang X, Li J, Xu Y, Shi J. The Masquelet technique triggers the formation of a network involving LncRNA, circRNA, miRNA, and mRNA during bone repair. Ann Med 2024; 56:2395591. [PMID: 39444146 PMCID: PMC11504341 DOI: 10.1080/07853890.2024.2395591] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Revised: 03/26/2024] [Accepted: 03/26/2024] [Indexed: 10/25/2024] Open
Abstract
BACKGROUND The ceRNA network, which is competitive endogenous RNA, uncovers a fresh mechanism of RNA interaction and holds significant importance in diverse biological processes. The aim of this study is to investigate the molecular process of induced membrane (IM) formation in bone defects using the Masquelet's induced membrane technique (MIMT), in order to offer novel insights and a theoretical foundation for enhancing the treatment of bone defects with MIMT. METHODS In this work, we identified differentially expressed mRNAs (DEGs), lncRNAs (DELs), circRNAs (DECs), and miRNAs (DEMs). To explore the primary functions of the shared DEGs, we utilized Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, predictions were made for lncRNA-miRNA and miRNA-mRNA interactions, and the Cytoscape software was utilized to construct the regulatory network for ceRNA. RESULTS By integrating GO and KEGG enrichment analysis, a total of 385 differentially expressed genes (DEGs) were discovered in the samples from the MIMT-treated group. Additionally, after re-annotating the probes and intersecting two sets of differently expressed miRNAs, 1304 differentially expressed lncRNAs (DELs) and 23 differentially expressed circRNAs (DECs) were identified. Furthermore, 13 differentially expressed miRNAs (DEMs) were obtained. Moreover, utilizing the anticipated objectives of DEMs, we acquired 1203 pairs of lncRNA-miRNA-mRNA interactors (comprising 24 lncRNAs, 10 miRNAs, and 115 mRNAs) and 250 pairs of circRNA-miRNA-mRNA interactions (comprising 7 circRNAs, 9 miRNAs, and 115 mRNAs). CEBPA, DGAT2, CDKN1A, PLIN2, and CIDEC were identified as the five hub proteins in the PPI network. LncRNA/circRNA-hsa-miR-671-5p could potentially regulate the primary central protein, CEBPA. CONCLUSIONS In this study, we described the potential regulatory mechanism of the MIMT in treating bone defects. We proposed a new lncRNA-miRNA-mRNA ceRNA network that could help further explore the molecular mechanisms of bone repair.
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Affiliation(s)
- Muguo Song
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
| | - Xiaoyong Yang
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
| | - Xijiao Zhang
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
| | - Junyi Li
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
| | - Yongqing Xu
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
| | - Jian Shi
- Department of Orthopaedics, 920th Hospital of the Joint Logistics Support Force of the PLA, Kunming City, Yunnan Province, China
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Herb M, Schatz V, Hadrian K, Hos D, Holoborodko B, Jantsch J, Brigo N. Macrophage variants in laboratory research: most are well done, but some are RAW. Front Cell Infect Microbiol 2024; 14:1457323. [PMID: 39445217 PMCID: PMC11496307 DOI: 10.3389/fcimb.2024.1457323] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2024] [Accepted: 09/06/2024] [Indexed: 10/25/2024] Open
Abstract
Macrophages play a pivotal role in the innate immune response. While their most characteristic function is phagocytosis, it is important not to solely characterize macrophages by this activity. Their crucial roles in body development, homeostasis, repair, and immune responses against pathogens necessitate a broader understanding. Macrophages exhibit remarkable plasticity, allowing them to modify their functional characteristics in response to the tissue microenvironment (tissue type, presence of pathogens or inflammation, and specific signals from neighboring cells) swiftly. While there is no single defined "macrophage" entity, there is a diverse array of macrophage types because macrophage ontogeny involves the differentiation of progenitor cells into tissue-resident macrophages, as well as the recruitment and differentiation of circulating monocytes in response to tissue-specific cues. In addition, macrophages continuously sense and respond to environmental cues and tissue conditions, adjusting their functional and metabolic states accordingly. Consequently, it is of paramount importance to comprehend the heterogeneous origins and functions of macrophages employed in in vitro studies, as each available in vitro macrophage model is associated with specific sets of strengths and limitations. This review centers its attention on a comprehensive comparison between immortalized mouse macrophage cell lines and primary mouse macrophages. It provides a detailed analysis of the strengths and weaknesses inherent in these in vitro models. Finally, it explores the subtle distinctions between diverse macrophage cell lines, offering insights into numerous factors beyond the model type that can profoundly influence macrophage function.
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Affiliation(s)
- Marc Herb
- Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Valentin Schatz
- Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Karina Hadrian
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Deniz Hos
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Bohdan Holoborodko
- Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg and University of Regensburg, Regensburg, Germany
| | - Jonathan Jantsch
- Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Natascha Brigo
- Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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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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Juncker T, Chatton B, Donzeau M. The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection. Cells 2023; 12:1591. [PMID: 37371061 DOI: 10.3390/cells12121591] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Revised: 05/31/2023] [Accepted: 06/07/2023] [Indexed: 06/29/2023] Open
Abstract
Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed VHHs and the antigen present in the cell, preventing the necessity to remove the excess unbound VHHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.
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Affiliation(s)
- Théo Juncker
- UMR7242 Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, F-67412 Illkirch, France
| | - Bruno Chatton
- UMR7242 Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, F-67412 Illkirch, France
| | - Mariel Donzeau
- UMR7242 Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, F-67412 Illkirch, France
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Zenin V, Tsedilin A, Yurkova M, Siniavin A, Fedorov A. Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities. PLoS One 2023; 18:e0286752. [PMID: 37289764 PMCID: PMC10249821 DOI: 10.1371/journal.pone.0286752] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Accepted: 05/23/2023] [Indexed: 06/10/2023] Open
Abstract
Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 μM while the standard one had 0.0180 μM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.
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Affiliation(s)
- Vladimir Zenin
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation
| | - Andrey Tsedilin
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation
| | - Maria Yurkova
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation
| | - Andrey Siniavin
- Ivanovsky Institute of Virology, N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russian Federation
| | - Alexey Fedorov
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation
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Chaudhary S, Ali Z, Tehseen M, Haney EF, Pantoja-Angles A, Alshehri S, Wang T, Clancy GJ, Ayach M, Hauser C, Hong PY, Hamdan SM, Hancock REW, Mahfouz M. Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens. Nat Commun 2023; 14:1464. [PMID: 36928189 PMCID: PMC10020429 DOI: 10.1038/s41467-023-37003-z] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Accepted: 02/27/2023] [Indexed: 03/18/2023] Open
Abstract
Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.
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Affiliation(s)
- Shahid Chaudhary
- Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Zahir Ali
- Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Muhammad Tehseen
- Laboratory of DNA Replication and Recombination, Division of Biological Sciences and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Evan F Haney
- Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada
| | - Aarón Pantoja-Angles
- Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Salwa Alshehri
- Laboratory for Nanomedicine, Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
- Biochemistry Department, Faculty of Science, University of Jeddah, Jeddah, 21577, Saudi Arabia
| | - Tiannyu Wang
- Water Desalination and Reuse Center, Division of Biological Sciences and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Gerard J Clancy
- Analytical Chemistry Core Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia
| | - Maya Ayach
- Imaging & Characterization Core Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia
| | - Charlotte Hauser
- Laboratory for Nanomedicine, Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Pei-Ying Hong
- Water Desalination and Reuse Center, Division of Biological Sciences and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Samir M Hamdan
- Laboratory of DNA Replication and Recombination, Division of Biological Sciences and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
| | - Robert E W Hancock
- Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada
| | - Magdy Mahfouz
- Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia.
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Chinnadurai R. Advanced Technologies for Potency Assay Measurement. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1420:81-95. [PMID: 37258785 DOI: 10.1007/978-3-031-30040-0_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
Crucial for their application, cell products need to be well-characterized in the cell manufacturing facilities and conform to regulatory approval criteria before infusion into the patients. Mesenchymal Stromal Cells (MSCs) are the leading cell therapy candidate in clinical trials worldwide. Early phase clinical trials have demonstrated that MSCs display an excellent safety profile and are well tolerated. However, MSCs have also exhibited contradictory efficacy in later-phase clinical trials with reasons for this discrepancy including poorly understood mechanism of MSC therapeutic action. With likelihood that a number of attributes are involved in MSC derived clinical benefit, an assay that measures a single quality of may not adequately reflect potency, thus a combination of bioassays and analytical methods, collectively called "assay matrix" are favoured for defining the potency of MSC more adequately. This chapter highlights advanced technologies and targets that can achieve quantitative measurement for a range of MSC attributes, including immunological, genomic, secretome, phosphorylation, morphological, biomaterial, angiogenic and metabolic assays.
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Affiliation(s)
- Raghavan Chinnadurai
- Department of Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA.
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Gastrointestinal Tract Stabilized Protein Delivery Using Disulfide Thermostable Exoshell System. Int J Mol Sci 2022; 23:ijms23179856. [PMID: 36077259 PMCID: PMC9456531 DOI: 10.3390/ijms23179856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2022] [Revised: 08/12/2022] [Accepted: 08/13/2022] [Indexed: 11/17/2022] Open
Abstract
Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.
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10
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Soni AP, Lee J, Shin K, Koiwa H, Hwang I. Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana. FRONTIERS IN PLANT SCIENCE 2022; 13:922694. [PMID: 35712604 PMCID: PMC9197560 DOI: 10.3389/fpls.2022.922694] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2022] [Accepted: 05/11/2022] [Indexed: 06/15/2023]
Abstract
The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFβ1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.
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Affiliation(s)
- Aditya Prakash Soni
- Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea
| | - Juhee Lee
- Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea
- Department of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South Korea
| | - Kunyoo Shin
- Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea
- Department of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South Korea
| | - Hisashi Koiwa
- Molecular and Environmental Plant Sciences, Texas A&M University, College Station, TX, United States
- Vegetable and Fruit Development Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX, United States
| | - Inhwan Hwang
- Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea
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11
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Fus-Kujawa A, Prus P, Bajdak-Rusinek K, Teper P, Gawron K, Kowalczuk A, Sieron AL. An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Front Bioeng Biotechnol 2021; 9:701031. [PMID: 34354988 PMCID: PMC8330802 DOI: 10.3389/fbioe.2021.701031] [Citation(s) in RCA: 90] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Accepted: 06/28/2021] [Indexed: 11/13/2022] Open
Abstract
Transfection is a powerful analytical tool enabling studies of gene products and functions in eukaryotic cells. Successful delivery of genetic material into cells depends on DNA quantity and quality, incubation time and ratio of transfection reagent to DNA, the origin, type and the passage of transfected cells, and the presence or absence of serum in the cell culture. So far a number of transfection methods that use viruses, non-viral particles or physical factors as the nucleic acids carriers have been developed. Among non-viral carriers, the cationic polymers are proposed as the most attractive ones due to the possibility of their chemical structure modification, low toxicity and immunogenicity. In this review the delivery systems as well as physical, biological and chemical methods used for eukaryotic cells transfection are described and discussed.
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Affiliation(s)
- Agnieszka Fus-Kujawa
- Department of Molecular Biology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland
| | - Pawel Prus
- Department of Molecular Biology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland
- Students’ Scientific Society, Katowice, Poland
| | - Karolina Bajdak-Rusinek
- Department of Medical Genetics, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland
| | - Paulina Teper
- Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland
| | - Katarzyna Gawron
- Department of Molecular Biology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland
| | - Agnieszka Kowalczuk
- Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland
| | - Aleksander L. Sieron
- Department of Molecular Biology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland
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12
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Nomura Y, Yamamura J, Fukui C, Fujimaki H, Sakamoto K, Matsuo KI, Kuromatsu H, Kikuchi Y, Haishima Y. Performance evaluation of bactericidal effect and endotoxin inactivation by low-temperature ozone/hydrogen peroxide mixed gas exposure. J Biomed Mater Res B Appl Biomater 2021; 109:1807-1816. [PMID: 33783121 DOI: 10.1002/jbm.b.34840] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 03/15/2021] [Accepted: 03/19/2021] [Indexed: 11/09/2022]
Abstract
This study evaluated the performance of a new O3 /H2 O2 mixed gas sterilization instrument for killing microorganisms and inactivating bacterial endotoxin at low temperatures. Sterility assurance level was achieved by an over 6-log reduction of Geobacillus stearothermophilus ATCC 12980, and the decimal reduction value was 0.77 min in sterilization mode. A reduction of over 3 logs in Limulus amebocyte lysate coagulation activity of purified endotoxin from Escherichia coli was observed after treatment in endotoxin-inactivation mode. The same inactivation ability was observed when treating dried bacterial cells. Biomaterials made of polymer or metal did not exhibit cytotoxicity after gas exposure at O3 concentrations below 200 ppm. As the results of human cell-based pyrogen testing, significant amounts of endotoxin that were over the limit for medical devices contacting cerebrospinal fluid (2.15 EU/device) were detected on scissors washed with a washer-disinfector and sterilized with ethylene oxide or autoclaving. In contrast, endotoxin decreased to 0.29 ± 0.05 EU/device after O3 /H2 O2 mixed gas sterilization in endotoxin-inactivation mode. Compared to conventional gas sterilization methods, O3 /H2 O2 mixed gas has high sterilization ability and a strong capacity to inactivate endotoxin. It is expected that this sterilization technology will improve the safety of reusable medical devices and utensils for regenerative medicine.
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Affiliation(s)
- Yusuke Nomura
- Division of Medical Devices, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
| | - Junji Yamamura
- Environment and Cleran Group, Strategy Development Department, Industrial Systems and General-Purpose Machinery Business Area, IHI Corporation, Tokyo, Japan
| | - Chie Fukui
- Division of Medical Devices, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
| | - Hideo Fujimaki
- Research Department, Public Welfare Institute of Scientific Research Foundation, Tokyo, Japan
| | - Kazuyuki Sakamoto
- Environment and Cleran Group, Strategy Development Department, Industrial Systems and General-Purpose Machinery Business Area, IHI Corporation, Tokyo, Japan
| | - Ken-Ichi Matsuo
- Environment and Cleran Group, Strategy Development Department, Industrial Systems and General-Purpose Machinery Business Area, IHI Corporation, Tokyo, Japan
| | - Hisashi Kuromatsu
- Environment and Cleran Group, Strategy Development Department, Industrial Systems and General-Purpose Machinery Business Area, IHI Corporation, Tokyo, Japan
| | - Yutaka Kikuchi
- Division of Microbiology, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
| | - Yuji Haishima
- Division of Medical Devices, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
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13
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Diaz Diaz AC, Shearer JA, Malone K, Waeber C. Acute Treatment With Fingolimod Does Not Confer Long-Term Benefit in a Mouse Model of Intracerebral Haemorrhage. Front Pharmacol 2021; 11:613103. [PMID: 33488389 PMCID: PMC7821021 DOI: 10.3389/fphar.2020.613103] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 11/23/2020] [Indexed: 01/01/2023] Open
Abstract
Intracerebral haemorrhage (ICH) has no specific treatment, but accounts for up to 15% of all strokes and has the highest mortality. Fingolimod (FTY720) is an immunomodulator approved for the management of multiple sclerosis, with abundant evidence of efficacy in experimental ischemic stroke, and more limited evidence in experimental ICH. The goal of this study was to confirm the efficacy of fingolimod in experimental ICH using rigorous and statistically well-powered studies. ICH was induced in C57BL/6JOlaHsd male and female mice by intrastriatal bacterial collagenase injection. Fingolimod (0.5 mg/kg) or saline was administered intraperitoneally after 0.5, 24 and 72 h, in a randomized and blinded manner. Functional improvement with cylinder, wire hanging, and foot fault tests was evaluated one and two weeks later. Lesion volume and hemispheric atrophy were quantified at the 14-day endpoint. There was a higher mortality in saline-treated females compared to fingolimod-treated females and saline-treated males. There was no treatment- or gender-related difference in the behavioural tests. Histological outcome measures did not differ between any of the groups. These results, contrasting with those of previous studies of fingolimod in experimental ICH, emphasize the importance of rigorous testing of this agent in models more representative of the clinical situation.
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Affiliation(s)
| | | | - Kyle Malone
- School of Pharmacy, University College Cork, Cork, Ireland
| | - Christian Waeber
- School of Pharmacy, University College Cork, Cork, Ireland.,Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland
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14
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Zhao G, Ge Y, Zhang C, Zhang L, Xu J, Qi L, Li W. Progress of Mesenchymal Stem Cell-Derived Exosomes in Tissue Repair. Curr Pharm Des 2020; 26:2022-2037. [PMID: 32310043 DOI: 10.2174/1381612826666200420144805] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2019] [Accepted: 03/25/2020] [Indexed: 12/17/2022]
Abstract
Mesenchymal stem cells (MSCs) are a kind of adult stem cells with self-replication and multidirectional differentiation, which can differentiate into tissue-specific cells under physiological conditions, maintaining tissue self-renewal and physiological functions. They play a role in the pathological condition by lateral differentiation into tissue-specific cells, replacing damaged tissue cells by playing the role of a regenerative medicine , or repairing damaged tissues through angiogenesis, thereby, regulating immune responses, inflammatory responses, and inhibiting apoptosis. It has become an important seed cell for tissue repair and organ reconstruction, and cell therapy based on MSCs has been widely used clinically. The study found that the probability of stem cells migrating to the damaged area after transplantation or differentiating into damaged cells is very low, so the researchers believe the leading role of stem cell transplantation for tissue repair is paracrine secretion, secreting growth factors, cytokines or other components. Exosomes are biologically active small vesicles secreted by MSCs. Recent studies have shown that they can transfer functional proteins, RNA, microRNAs, and lncRNAs between cells, and greatly reduce the immune response. Under the premise of promoting proliferation and inhibition of apoptosis, they play a repair role in tissue damage, which is caused by a variety of diseases. In this paper, the biological characteristics of exosomes (MSCs-exosomes) derived from mesenchymal stem cells, intercellular transport mechanisms, and their research progress in the field of stem cell therapy are reviewed.
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Affiliation(s)
- Guifang Zhao
- School of Basic Medical Sciences, Jilin Medical University, Jilin 132013, China.,Qingyuan People's Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan 511518, Guangzhou Province, China
| | - Yiwen Ge
- School of Basic Medical Sciences, Jilin Medical University, Jilin 132013, China
| | - Chenyingnan Zhang
- School of Basic Medical Sciences, Jilin Medical University, Jilin 132013, China
| | - Leyi Zhang
- School of Pharmacy, Jilin Medical University, Jilin 132013, China
| | - Junjie Xu
- School of Basic Medical Sciences, Jilin Medical University, Jilin 132013, China
| | - Ling Qi
- Qingyuan People's Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan 511518, Guangzhou Province, China.,School of Basic Medical Sciences, Department of Pathophysiology, Jilin Medical University, Jilin 132013, China
| | - Wenliang Li
- School of Pharmacy, Jilin Medical University, Jilin 132013, China
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15
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Groen WMGAC, Utomo L, Castilho M, Gawlitta D, Malda J, van Weeren PR, Levato R, Korthagen NM. Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro. Int J Mol Sci 2020; 21:E8571. [PMID: 33202964 PMCID: PMC7696312 DOI: 10.3390/ijms21228571] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Revised: 11/02/2020] [Accepted: 11/11/2020] [Indexed: 12/31/2022] Open
Abstract
Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/mL and three with <10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-α and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.
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Affiliation(s)
- Wilhelmina M. G. A. C. Groen
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.M.); (R.L.); (N.M.K.)
- Department of Orthopaedics, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands;
| | - Lizette Utomo
- Department of Oral and Maxillofacial Surgery and Special Dental Care, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands; (L.U.); (D.G.)
| | - Miguel Castilho
- Department of Orthopaedics, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands;
| | - Debby Gawlitta
- Department of Oral and Maxillofacial Surgery and Special Dental Care, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands; (L.U.); (D.G.)
| | - Jos Malda
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.M.); (R.L.); (N.M.K.)
- Department of Orthopaedics, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands;
| | - P. René van Weeren
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.M.); (R.L.); (N.M.K.)
| | - Riccardo Levato
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.M.); (R.L.); (N.M.K.)
- Department of Orthopaedics, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands;
| | - Nicoline M. Korthagen
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.M.); (R.L.); (N.M.K.)
- Department of Orthopaedics, Regenerative Medicine Center, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands;
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16
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Sah SK, Agrahari G, Kim TY. Insights into superoxide dismutase 3 in regulating biological and functional properties of mesenchymal stem cells. Cell Biosci 2020; 10:22. [PMID: 32128111 PMCID: PMC7045732 DOI: 10.1186/s13578-020-00386-3] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Accepted: 02/14/2020] [Indexed: 12/11/2022] Open
Abstract
Mesenchymal stem cells (MSCs) have been extensively studied and implicated for the cell-based therapy in several diseases due to theirs immunomodulatory properties. Embryonic stem cells and induced-pluripotent stem cells have either ethical issues or concerns regarding the formation of teratomas, introduction of mutations into genome during prolonged culture, respectively which limit their uses in clinical settings. On the other hand, MSCs also encounter certain limitation of circumscribed survival and reduced immunomodulatory potential during transplantation. Plethora of research is undergoing to improve the efficacy of MSCs during therapy. Several compounds and novel techniques have been employed to increase the therapeutic potency of MSCs. MSCs secreted superoxide dismutase 3 (SOD3) may be the mechanism for exhibiting direct antioxidant activities by MSCs. SOD3 is a well known antioxidant enzyme and recently known to possess immunomodulatory properties. Along with superoxide scavenging property, SOD3 also displays anti-angiogenic, anti-chemotactic and anti-inflammatory functions in both enzymatic and non-enzymatic manners. In this review, we summarize the emerging role of SOD3 secreted from MSCs and SOD3’s effects during cell-based therapy.
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Affiliation(s)
- Shyam Kishor Sah
- 1Department of Reconstructive Sciences, Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CT 06032 USA.,2Laboratory of Dermato-immunology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul, 06591 Republic of Korea
| | - Gaurav Agrahari
- 2Laboratory of Dermato-immunology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul, 06591 Republic of Korea
| | - Tae-Yoon Kim
- 2Laboratory of Dermato-immunology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul, 06591 Republic of Korea
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17
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Islam MR, Choi S, Muthamilselvan T, Shin K, Hwang I. In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana. FRONTIERS IN PLANT SCIENCE 2020; 11:440. [PMID: 32328082 PMCID: PMC7160244 DOI: 10.3389/fpls.2020.00440] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Accepted: 03/25/2020] [Indexed: 05/22/2023]
Abstract
Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni2+-NTA affinity purification resulting in a yield of 32.49 mg/kg, and the N-terminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.
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Affiliation(s)
- Md Reyazul Islam
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, South Korea
| | - Seoyoung Choi
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South, Korea
| | - Thangarasu Muthamilselvan
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, South Korea
| | - Kunyoo Shin
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South, Korea
| | - Inhwan Hwang
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, South Korea
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South, Korea
- *Correspondence: Inhwan Hwang,
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18
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Islam MR, Kwak J, Lee J, Hong S, Khan MRI, Lee Y, Lee Y, Lee S, Hwang I. Cost-effective production of tag-less recombinant protein in Nicotiana benthamiana. PLANT BIOTECHNOLOGY JOURNAL 2019; 17:1094-1105. [PMID: 30468023 PMCID: PMC6523591 DOI: 10.1111/pbi.13040] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/13/2018] [Revised: 11/02/2018] [Accepted: 11/04/2018] [Indexed: 05/03/2023]
Abstract
Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost-effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost-effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose-binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin-related modifier (SUMO) and SUMO-specific protease were used to remove it. This method, together with size-exclusion chromatography, enabled purification of human interleukin-6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium-mediated transient expression. Plant-produced hIL6 (P-hIL6) contained less than 0.2 EU/μg (0.02 ng/mL) endotoxin. P-hIL6 activated the Janus kinase-signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL-21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.
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Affiliation(s)
- Md Reyazul Islam
- Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangKorea
| | - Ju‐Won Kwak
- Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangKorea
| | - Jeon‐soo Lee
- Department of Life SciencePohang University of Science and TechnologyPohangKorea
| | - Sung‐Wook Hong
- Department of Life SciencePohang University of Science and TechnologyPohangKorea
| | - Md Rezaul Islam Khan
- Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangKorea
| | - Yongjik Lee
- Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangKorea
| | - Yoontae Lee
- Department of Life SciencePohang University of Science and TechnologyPohangKorea
| | - Seung‐Woo Lee
- Department of Life SciencePohang University of Science and TechnologyPohangKorea
| | - Inhwan Hwang
- Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangKorea
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19
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Nomura Y, Fukui C, Morishita Y, Haishima Y. A biological study establishing the endotoxin limit for osteoblast and adipocyte differentiation of human mesenchymal stem cells. Regen Ther 2018; 8:46-57. [PMID: 30271865 PMCID: PMC6149188 DOI: 10.1016/j.reth.2018.01.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Revised: 12/25/2017] [Accepted: 01/09/2018] [Indexed: 01/25/2023] Open
Abstract
INTRODUCTION Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions, such as hypoxia or inflammation after transplantation in vivo. Although endotoxin testing is required for evaluating the quality and safety of transplanted MSCs, no reports on their dose response to endotoxins are available to establish the limits for in vitro MSC culture systems. In the present study, we aimed to accurately quantify the risk of endotoxin contamination in cell culture systems to establish an acceptable endotoxin limit for the differentiation of hMSC osteoblasts and adipocytes. METHODS Three types of bone marrow-derived hMSCs (hMSC-1: 21-year-old, M/B; hMSC-2: 36-year-old, M/B; hMSC-3: 43-year-old, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in osteogenic or adipogenic differentiation media, respectively, from commercial kits, containing various concentrations of endotoxin (0.01-100 ng/ml). The degree of adipocyte and osteoblast differentiation was estimated by fluorescent staining of lipid droplets and hydroxyapatite, respectively. To clarify the molecular mechanism underlying the effect of endotoxin on hMSC differentiation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. RESULTS Although endotoxin did not effect the adipocyte differentiation of hMSCs, osteoblast differentiation was enhanced by various endotoxin concentrations: over 1 ng/ml, for hMSC-1; 10 ng/ml, for hMSC-2; and 100 ng/ml, for hMSC-3. Proteomic analysis of hMSC-1 cells revealed up-regulation of many proteins related to bone formation. These results suggested that endotoxin enhances the osteoblast differentiation of MSCs depending on the cell type. CONCLUSIONS Since endotoxins can affect various cellular functions, an endotoxin limit should be established for in vitro MSC cultures. Its no-observed-adverse-effect level was 0.1 ng/ml based on the effect on the hMSC osteoblast differentiation, but it may not necessarily be the limit for ADSCs.
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Affiliation(s)
| | | | | | - Yuji Haishima
- Division of Medical Devices, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
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