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Matiukhova M, Ryapolova A, Andriianov V, Reshetnikov V, Zhuravleva S, Ivanov R, Karabelsky A, Minskaia E. A comprehensive analysis of induced pluripotent stem cell (iPSC) production and applications. Front Cell Dev Biol 2025; 13:1593207. [PMID: 40406420 PMCID: PMC12095295 DOI: 10.3389/fcell.2025.1593207] [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: 03/13/2025] [Accepted: 04/14/2025] [Indexed: 05/26/2025] Open
Abstract
The ability to reprogram mature, differentiated cells into induced pluripotent stem cells (iPSCs) using exogenous pluripotency factors opened up unprecedented opportunities for their application in biomedicine. iPSCs are already successfully used in cell and regenerative therapy, as various drug discovery platforms and for in vitro disease modeling. However, even though already 20 years have passed since their discovery, the production of iPSC-based therapies is still associated with a number of hurdles due to low reprogramming efficiency, the complexity of accurate characterization of the resulting colonies, and the concerns associated with the safety of this approach. However, significant progress in many areas of molecular biology facilitated the production, characterization, and thorough assessment of the safety profile of iPSCs. The number of iPSC-based studies has been steadily increasing in recent years, leading to the accumulation of significant knowledge in this area. In this review, we aimed to provide a comprehensive analysis of methods used for reprogramming and subsequent characterization of iPSCs, discussed barriers towards achieving these goals, and various approaches to improve the efficiency of reprogramming of different cell populations. In addition, we focused on the analysis of iPSC application in preclinical and clinical studies. The accumulated breadth of data helps to draw conclusions about the future of this technology in biomedicine.
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Affiliation(s)
| | | | | | | | | | | | | | - Ekaterina Minskaia
- Translational Medicine Research Center, Sirius University of Science and Technology, Sochi, Russia
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2
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Wang AYL, Chang YC, Chen KH, Loh CYY. Potential Application of Modified mRNA in Cardiac Regeneration. Cell Transplant 2024; 33:9636897241248956. [PMID: 38715279 PMCID: PMC11080755 DOI: 10.1177/09636897241248956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Revised: 03/26/2024] [Accepted: 04/07/2024] [Indexed: 05/12/2024] Open
Abstract
Heart failure remains the leading cause of human death worldwide. After a heart attack, the formation of scar tissue due to the massive death of cardiomyocytes leads to heart failure and sudden death in most cases. In addition, the regenerative ability of the adult heart is limited after injury, partly due to cell-cycle arrest in cardiomyocytes. In the current post-COVID-19 era, urgently authorized modified mRNA (modRNA) vaccines have been widely used to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Therefore, modRNA-based protein replacement may act as an alternative strategy for improving heart disease. It is a safe, effective, transient, low-immunogenic, and integration-free strategy for in vivo protein expression, in addition to recombinant protein and stem-cell regenerative therapies. In this review, we provide a summary of various cardiac factors that have been utilized with the modRNA method to enhance cardiovascular regeneration, cardiomyocyte proliferation, fibrosis inhibition, and apoptosis inhibition. We further discuss other cardiac factors, modRNA delivery methods, and injection methods using the modRNA approach to explore their application potential in heart disease. Factors for promoting cardiomyocyte proliferation such as a cocktail of three genes comprising FoxM1, Id1, and Jnk3-shRNA (FIJs), gp130, and melatonin have potential to be applied in the modRNA approach. We also discuss the current challenges with respect to modRNA-based cardiac regenerative medicine that need to be overcome to apply this approach to heart disease. This review provides a short description for investigators interested in the development of alternative cardiac regenerative medicines using the modRNA platform.
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Affiliation(s)
- Aline Yen Ling Wang
- Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Yun-Ching Chang
- Department of Health Industry Technology Management, Chung Shan Medical University, Taichung, Taiwan
- Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
| | - Kuan-Hung Chen
- Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital, Taoyuan, Taiwan
- College of Medicine, Chang Gung University, Taoyuan, Taiwan
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3
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Cho S, Aakash P, Lee S, Yoon YS. Endothelial cell direct reprogramming: Past, present, and future. J Mol Cell Cardiol 2023; 180:22-32. [PMID: 37080451 PMCID: PMC10330356 DOI: 10.1016/j.yjmcc.2023.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 04/04/2023] [Accepted: 04/17/2023] [Indexed: 04/22/2023]
Abstract
Ischemic cardiovascular disease still remains as a leading cause of morbidity and mortality despite various medical, surgical, and interventional therapy. As such, cell therapy has emerged as an attractive option because it tackles underlying problem of the diseases by inducing neovascularization in ischemic tissue. After overall failure of adult stem or progenitor cells, studies attempted to generate endothelial cells (ECs) from pluripotent stem cells (PSCs). While endothelial cells (ECs) differentiated from PSCs successfully induced vascular regeneration, differentiating volatility and tumorigenic potential is a concern for their clinical applications. Alternatively, direct reprogramming strategies employ lineage-specific factors to change cell fate without achieving pluripotency. ECs have been successfully reprogrammed via ectopic expression of transcription factors (TFs) from endothelial lineage. The reprogrammed ECs induced neovascularization in vitro and in vivo and thus demonstrated their therapeutic value in animal models of vascular insufficiency. Methods of delivering reprogramming factors include lentiviral or retroviral vectors and more clinically relevant, non-integrative adenoviral and episomal vectors. Most studies made use of fibroblast as a source cell for reprogramming, but reprogrammability of other clinically relevant source cell types has to be evaluated. Specific mechanisms and small molecules that are involved in the aforementioned processes tackles challenges associated with direct reprogramming efficiency and maintenance of reprogrammed EC characteristics. After all, this review provides summary of past and contemporary methods of direct endothelial reprogramming and discusses the future direction to overcome these challenges to acquire clinically applicable reprogrammed ECs.
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Affiliation(s)
- Seonggeon Cho
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Parthasarathy Aakash
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Sangho Lee
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA.
| | - Young-Sup Yoon
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of Korea.
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4
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Transition from Animal-Based to Human Induced Pluripotent Stem Cells (iPSCs)-Based Models of Neurodevelopmental Disorders: Opportunities and Challenges. Cells 2023; 12:cells12040538. [PMID: 36831205 PMCID: PMC9954744 DOI: 10.3390/cells12040538] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 01/25/2023] [Accepted: 02/02/2023] [Indexed: 02/11/2023] Open
Abstract
Neurodevelopmental disorders (NDDs) arise from the disruption of highly coordinated mechanisms underlying brain development, which results in impaired sensory, motor and/or cognitive functions. Although rodent models have offered very relevant insights to the field, the translation of findings to clinics, particularly regarding therapeutic approaches for these diseases, remains challenging. Part of the explanation for this failure may be the genetic differences-some targets not being conserved between species-and, most importantly, the differences in regulation of gene expression. This prompts the use of human-derived models to study NDDS. The generation of human induced pluripotent stem cells (hIPSCs) added a new suitable alternative to overcome species limitations, allowing for the study of human neuronal development while maintaining the genetic background of the donor patient. Several hIPSC models of NDDs already proved their worth by mimicking several pathological phenotypes found in humans. In this review, we highlight the utility of hIPSCs to pave new paths for NDD research and development of new therapeutic tools, summarize the challenges and advances of hIPSC-culture and neuronal differentiation protocols and discuss the best way to take advantage of these models, illustrating this with examples of success for some NDDs.
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5
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Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency. Pharmaceutics 2023; 15:pharmaceutics15010286. [PMID: 36678915 PMCID: PMC9861957 DOI: 10.3390/pharmaceutics15010286] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2022] [Revised: 12/31/2022] [Accepted: 01/09/2023] [Indexed: 01/19/2023] Open
Abstract
Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.
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6
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Bailly A, Milhavet O, Lemaitre JM. RNA-Based Strategies for Cell Reprogramming toward Pluripotency. Pharmaceutics 2022; 14:317. [PMID: 35214051 PMCID: PMC8876983 DOI: 10.3390/pharmaceutics14020317] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/16/2022] [Accepted: 01/25/2022] [Indexed: 02/04/2023] Open
Abstract
Cell therapy approaches to treat a wide range of pathologies have greatly benefited from cell reprogramming techniques that allow the conversion of a somatic cell into a pluripotent cell. Many technological developments have been made since the initial major discovery of this biological process. Recently reprogramming methods based on the use of RNA have emerged and seem very promising. Thus, in this review we will focus on presenting the interest of such methods for cell reprogramming but also how these RNA-based strategies can be extended to eventually lead to medical applications to improve healthspan and longevity.
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Affiliation(s)
- Anaëlle Bailly
- IRMB, University Montpellier, INSERM, 34295 Montpellier, France
- INGRAALYS, SA, IRMB, Incubator Cyborg, 34295 Montpellier, France
| | - Ollivier Milhavet
- IRMB, University Montpellier, INSERM, CNRS, 34295 Montpellier, France
- SAFE-iPSC Facility, CHU Montpellier, 34295 Montpellier, France
| | - Jean-Marc Lemaitre
- IRMB, University Montpellier, INSERM, 34295 Montpellier, France
- SAFE-iPSC Facility, CHU Montpellier, 34295 Montpellier, France
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7
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Yuen JSK, Stout AJ, Kawecki NS, Letcher SM, Theodossiou SK, Cohen JM, Barrick BM, Saad MK, Rubio NR, Pietropinto JA, DiCindio H, Zhang SW, Rowat AC, Kaplan DL. Perspectives on scaling production of adipose tissue for food applications. Biomaterials 2022; 280:121273. [PMID: 34933254 PMCID: PMC8725203 DOI: 10.1016/j.biomaterials.2021.121273] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Revised: 11/22/2021] [Accepted: 11/23/2021] [Indexed: 01/03/2023]
Abstract
With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.
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Affiliation(s)
- John S K Yuen
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Andrew J Stout
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - N Stephanie Kawecki
- Department of Bioengineering, University of California Los Angeles, 410 Westwood Plaza, Los Angeles, CA, 90095, USA; Department of Integrative Biology & Physiology, University of California Los Angeles, Terasaki Life Sciences Building, 610 Charles E. Young Drive South, Los Angeles, CA, 90095, USA
| | - Sophia M Letcher
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Sophia K Theodossiou
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Julian M Cohen
- W. M. Keck Science Department, Pitzer College, 925 N Mills Ave, Claremont, CA, 91711, USA
| | - Brigid M Barrick
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Michael K Saad
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Natalie R Rubio
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Jaymie A Pietropinto
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Hailey DiCindio
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Sabrina W Zhang
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA
| | - Amy C Rowat
- Department of Bioengineering, University of California Los Angeles, 410 Westwood Plaza, Los Angeles, CA, 90095, USA; Department of Integrative Biology & Physiology, University of California Los Angeles, Terasaki Life Sciences Building, 610 Charles E. Young Drive South, Los Angeles, CA, 90095, USA
| | - David L Kaplan
- Biomedical Engineering Department, Tissue Engineering Resource Center, Tufts University, 4 Colby St, Medford, MA, 02155, USA.
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8
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Oswald A, Chakraborty A, Ni Y, Wettengel JM, Urban S, Protzer U. Concentration of Na +-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus. Sci Rep 2021; 11:19799. [PMID: 34611272 PMCID: PMC8492621 DOI: 10.1038/s41598-021-99263-3] [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: 04/07/2021] [Accepted: 09/21/2021] [Indexed: 02/07/2023] Open
Abstract
Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.
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Affiliation(s)
- Andreas Oswald
- Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany
| | - Anindita Chakraborty
- Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany
| | - Yi Ni
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany
| | - Jochen M Wettengel
- Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany
| | - Ulrike Protzer
- Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany.
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany.
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9
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Wang AYL. Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate. Int J Mol Sci 2021; 22:8148. [PMID: 34360910 PMCID: PMC8348611 DOI: 10.3390/ijms22158148] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 07/25/2021] [Accepted: 07/27/2021] [Indexed: 02/07/2023] Open
Abstract
Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.
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Affiliation(s)
- Aline Yen Ling Wang
- Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
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10
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Cho JY, Bhowmik P, Polowick PL, Dodard SG, El-Bakkari M, Nowak G, Fenniri H, Hemraz UD. Cellular Delivery of Plasmid DNA into Wheat Microspores Using Rosette Nanotubes. ACS OMEGA 2020; 5:24422-24433. [PMID: 33015458 PMCID: PMC7528298 DOI: 10.1021/acsomega.0c02830] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/14/2020] [Accepted: 09/02/2020] [Indexed: 06/11/2023]
Abstract
Plant genetic engineering offers promising solutions to the increasing demand for efficient, sustainable, and high-yielding crop production as well as changing environmental conditions. The main challenge for gene delivery in plants is the presence of a cell wall that limits the transportation of genes within the cells. Microspores are plant cells that are, under the right conditions, capable of generating embryos, leading to the formation of haploid plants. Here, we designed cationic and fluorescent rosette nanotubes (RNTs) that penetrate the cell walls of viable wheat microspores under mild conditions and in the absence of an external force. These nanomaterials can capture plasmid DNA to form RNT-DNA complexes and transport their DNA cargo into live microspores. The nanomaterials and the complexes formed were nontoxic to the microspores.
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Affiliation(s)
- Jae-Young Cho
- Nanotechnology Research Centre, National Research Council of Canada, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 2M9, Canada
| | - Pankaj Bhowmik
- Aquatic and Crop Resource Development, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada
| | - Patricia L Polowick
- Aquatic and Crop Resource Development, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada
| | - Sabine G Dodard
- Aquatic and Crop Resource Development, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
| | - Mounir El-Bakkari
- Nanotechnology Research Centre, National Research Council of Canada, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 2M9, Canada
| | - Goska Nowak
- Aquatic and Crop Resource Development, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada
| | - Hicham Fenniri
- Nanotechnology Research Centre, National Research Council of Canada, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 2M9, Canada
- Departments of Chemical, Biomedical Engineering, Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States
| | - Usha D Hemraz
- Aquatic and Crop Resource Development, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
- Nanotechnology Research Centre, National Research Council of Canada, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 2M9, Canada
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Borgohain MP, Haridhasapavalan KK, Dey C, Adhikari P, Thummer RP. An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications. Stem Cell Rev Rep 2020; 15:286-313. [PMID: 30417242 DOI: 10.1007/s12015-018-9861-6] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.
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Affiliation(s)
- Manash P Borgohain
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Krishna Kumar Haridhasapavalan
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Chandrima Dey
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Poulomi Adhikari
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
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12
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Reprogramming and transdifferentiation - two key processes for regenerative medicine. Eur J Pharmacol 2020; 882:173202. [PMID: 32562801 DOI: 10.1016/j.ejphar.2020.173202] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Revised: 04/22/2020] [Accepted: 05/13/2020] [Indexed: 12/11/2022]
Abstract
Regenerative medicine based on transplants obtained from donors or foetal and new-born mesenchymal stem cells, encounter important obstacles such as limited availability of organs, ethical issues and immune rejection. The growing demand for therapeutic methods for patients being treated after serious accidents, severe organ dysfunction and an increasing number of cancer surgeries, exceeds the possibilities of the therapies that are currently available. Reprogramming and transdifferentiation provide powerful bioengineering tools. Both procedures are based on the somatic differentiated cells, which are easily and unlimitedly available, like for example: fibroblasts. During the reprogramming procedure mature cells are converted into pluripotent cells - which are capable to differentiate into almost any kind of desired cells. Transdifferentiation directly converts differentiated cells of one type into another differentiated cells type. Both procedures allow to obtained patient's dedicated cells for therapeutic purpose in regenerative medicine. In combination with biomaterials, it is possible to obtain even whole anatomical structures. Those patient's dedicated structures may serve for them upon serious accidents with massive tissue damage but also upon cancer surgeries as a replacement of damaged organ. Detailed information about reprogramming and transdifferentiation procedures as well as the current state of the art are presented in our review.
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Rai N, Singh AK, Singh SK, Gaurishankar B, Kamble SC, Mishra P, Kotiya D, Barik S, Atri N, Gautam V. Recent technological advancements in stem cell research for targeted therapeutics. Drug Deliv Transl Res 2020; 10:1147-1169. [DOI: 10.1007/s13346-020-00766-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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14
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Abstract
Chimeric antigen receptor (CAR) cancer immunotherapy uses autologous immune system's cells, genetically modified, to reinforce the immune system against cancer cells. Genetic modification is usually mediated via viral transfection, despite the risk of insertional oncogenesis and off target side effects. In vitro-transcribed (IVT)-mRNA-mediated transfection could contribute to a much safer CAR therapy, since IVT-mRNA leaves no ultimate genetic residue in recipient cells. In this chapter, the IVT-mRNA generation procedure is described, from the selection of the target of the CAR T-cells, the cloning of the template for the in vitro transcription and the development of several chemical modifications for optimizing the structure and thus the stability of the produced CAR IVT-mRNA molecules. Among various transfection methods to efficiently express the CAR molecule on T-cells' surface, the electroporation and the cationic-lipid mediated transfection of the CAR IVT-mRNAs are described.
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Affiliation(s)
- Androulla N Miliotou
- Laboratory of Pharmacology, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Macedonia, Greece
| | - Lefkothea C Papadopoulou
- Laboratory of Pharmacology, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Macedonia, Greece.
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15
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Qadir MI, Bukhat S, Rasul S, Manzoor H, Manzoor M. RNA therapeutics: Identification of novel targets leading to drug discovery. J Cell Biochem 2019; 121:898-929. [DOI: 10.1002/jcb.29364] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 08/20/2019] [Indexed: 12/23/2022]
Affiliation(s)
- Muhammad Imran Qadir
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Sherien Bukhat
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Sumaira Rasul
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Hamid Manzoor
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Majid Manzoor
- College of Pharmaceutical Sciences Zhejiang University Hangzhou China
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16
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Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA. Stem Cells Int 2019; 2019:7641767. [PMID: 31320906 PMCID: PMC6607707 DOI: 10.1155/2019/7641767] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Accepted: 05/20/2019] [Indexed: 12/19/2022] Open
Abstract
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance in the fields of regenerative medicine, tissue engineering, and disease modeling. Patient-specific iPSCs have as an unlimited cell source a tremendous potential for generating various types of autologous cells. For the future clinical applicability of these iPSC-derived cells, the generation of iPSCs via nongenome integrating methods and the efficient reprogramming of patients' somatic cells are required. In this study, 2 different RNA-based footprint-free methods for the generation of iPSCs were compared: the use of synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors and GFP. Using both RNA-based methods, integration-free iPSCs without genomic alterations were obtained. The pluripotency characteristics identified by specific marker detection and the in vitro and in vivo trilineage differentiation capacity were comparable. Moreover, the incorporation of a GFP encoding sequence into the srRNA enabled a direct and convenient monitoring of the reprogramming procedure and the successful detection of srRNA translation in the transfected cells. Nevertheless, the use of a single srRNA to induce pluripotency was less time consuming, faster, and more efficient than the daily transfection of cells with synthetic mRNAs. Therefore, we believe that the srRNA-based approach might be more appropriate and efficient for the reprogramming of different types of somatic cells for clinical applications.
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17
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Recent Updates on Induced Pluripotent Stem Cells in Hematological Disorders. Stem Cells Int 2019; 2019:5171032. [PMID: 31191673 PMCID: PMC6525795 DOI: 10.1155/2019/5171032] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2018] [Accepted: 03/31/2019] [Indexed: 02/07/2023] Open
Abstract
Over the past decade, enormous progress has been made in the field of induced pluripotent stem cells (iPSCs). Patients' somatic cells such as skin fibroblasts or blood cells can be used to generate disease-specific pluripotent stem cells, which have unlimited proliferation and can differentiate into all cell types of the body. Human iPSCs offer great promises and opportunities for treatments of degenerative diseases and studying disease pathology and drug screening. So far, many iPSC-derived disease models have led to the discovery of novel pathological mechanisms as well as new drugs in the pipeline that have been tested in the iPSC-derived cells for efficacy and potential toxicities. Furthermore, recent advances in genome editing technology in combination with the iPSC technology have provided a versatile platform for studying stem cell biology and regenerative medicine. In this review, an overview of iPSCs, patient-specific iPSCs for disease modeling and drug screening, applications of iPSCs and genome editing technology in hematological disorders, remaining challenges, and future perspectives of iPSCs in hematological diseases will be discussed.
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18
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Vizirianakis IS, Miliotou AN, Mystridis GA, Andriotis EG, Andreadis II, Papadopoulou LC, Fatouros DG. Tackling pharmacological response heterogeneity by PBPK modeling to advance precision medicine productivity of nanotechnology and genomics therapeutics. EXPERT REVIEW OF PRECISION MEDICINE AND DRUG DEVELOPMENT 2019. [DOI: 10.1080/23808993.2019.1605828] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Ioannis S. Vizirianakis
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Androulla N. Miliotou
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - George A. Mystridis
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Eleftherios G. Andriotis
- Laboratory of Pharmaceutical Technology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Ioannis I. Andreadis
- Laboratory of Pharmaceutical Technology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Lefkothea C. Papadopoulou
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Dimitrios G. Fatouros
- Laboratory of Pharmaceutical Technology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece
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19
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Warren L, Lin C. mRNA-Based Genetic Reprogramming. Mol Ther 2019; 27:729-734. [PMID: 30598301 PMCID: PMC6453511 DOI: 10.1016/j.ymthe.2018.12.009] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Revised: 12/07/2018] [Accepted: 12/08/2018] [Indexed: 01/12/2023] Open
Abstract
The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient's own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several "non-integrating" reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously "footprint-free," most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic.
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Affiliation(s)
- Luigi Warren
- Cellular Reprogramming, Inc., Pasadena, CA, USA.
| | - Cory Lin
- Cellular Reprogramming, Inc., Pasadena, CA, USA
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20
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Badieyan ZS, Evans T. Concise Review: Application of Chemically Modified mRNA in Cell Fate Conversion and Tissue Engineering. Stem Cells Transl Med 2019; 8:833-843. [PMID: 30891922 PMCID: PMC6646692 DOI: 10.1002/sctm.18-0259] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2018] [Accepted: 01/27/2019] [Indexed: 12/19/2022] Open
Abstract
Chemically modified RNA (cmRNA) has potential as a safe and efficient tool for nucleic acid‐based therapies and regenerative medicine. Modifications in the chemistry of mRNA can enhance stability, reduce immunogenicity, and thus facilitate mRNA‐based nucleic acid therapy, which eliminates risk of insertional mutagenesis. In addition to these valuable advantages, the mRNA‐based method showed significantly higher efficacy for reprogramming somatic cells to pluripotency compared with DNA‐ or protein‐based methods. These findings suggest cmRNA can provide a powerful and safe tool for cell programming and reprogramming. Delivery methods, particularly using lipid nanoparticles, provide strategies for cell and organ‐specific targeting. The present study comprehensively compares studies that have used cmRNAs for cell fate conversion and tissue engineering. The information should be useful for investigators looking to choose the most efficient and straightforward cmRNA‐based strategy and protocol for tissue engineering and regenerative medicine research. stem cells translational medicine2019;8:833&843
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Affiliation(s)
- Zohreh Sadat Badieyan
- Department of Surgery, Weill Cornell Medicine, Cornell University, New York, New York, USA
| | - Todd Evans
- Department of Surgery, Weill Cornell Medicine, Cornell University, New York, New York, USA
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21
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Abstract
Research on stem cells is one of the fastest growing areas of regenerative medicine that paves the way for a comprehensive solution to cell therapy. Today, stem cells are precious assets for generating different types of cells derived from either natural embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. The iPS technology can revolutionize the future of clinics by offering personalized medicine, which will provide the future treatment for curing untreatable diseases. Although iPS cell therapy is now at its infancy, promising research has motivated scientists to pursue this therapeutic approach. In this article, we provide information regarding similarities and differences between ES and iPS cells, and focus on the non-integrating methods of iPS generation via RNA molecules, especially microRNAs with an emphasis on the elucidation of their role and importance in pluripotency.
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Affiliation(s)
- Abbas Beh-Pajooh
- REBIRTH-Group Translational Hepatology and Stem Cell Biology, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Tobias Cantz
- REBIRTH-Group Translational Hepatology and Stem Cell Biology, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.,Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany
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22
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Ulasov AV, Rosenkranz AA, Sobolev AS. Transcription factors: Time to deliver. J Control Release 2017; 269:24-35. [PMID: 29113792 DOI: 10.1016/j.jconrel.2017.11.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 11/02/2017] [Accepted: 11/03/2017] [Indexed: 12/17/2022]
Abstract
Transcription factors (TFs) are at the center of the broad regulatory network orchestrating gene expression programs that elicit different biological responses. For a long time, TFs have been considered as potent drug targets due to their implications in the pathogenesis of a variety of diseases. At the same time, TFs, located at convergence points of cellular regulatory pathways, are powerful tools providing opportunities both for cell type change and for managing the state of cells. This task formulation requires the TF modulation problem to come to the fore. We review several ways to manage TF activity (small molecules, transfection, nanocarriers, protein-based approaches), analyzing their limitations and the possibilities to overcome them. Delivery of TFs could revolutionize the biomedical field. Whether this forecast comes true will depend on the ability to develop convenient technologies for targeted delivery of TFs.
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Affiliation(s)
- Alexey V Ulasov
- Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia
| | - Andrey A Rosenkranz
- Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia; Faculty of Biology, Moscow State University, 1-12 Leninskiye Gory St., 119234 Moscow, Russia
| | - Alexander S Sobolev
- Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia; Faculty of Biology, Moscow State University, 1-12 Leninskiye Gory St., 119234 Moscow, Russia.
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23
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Footprint-free human fetal foreskin derived iPSCs: A tool for modeling hepatogenesis associated gene regulatory networks. Sci Rep 2017; 7:6294. [PMID: 28740077 PMCID: PMC5524812 DOI: 10.1038/s41598-017-06546-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Accepted: 06/13/2017] [Indexed: 12/17/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general.
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24
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Kim SM, Lim MS, Lee EH, Jung SJ, Chung HY, Kim CH, Park CH. Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs. Mol Ther 2017; 25:2028-2037. [PMID: 28705346 DOI: 10.1016/j.ymthe.2017.06.015] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 06/15/2017] [Accepted: 06/18/2017] [Indexed: 12/25/2022] Open
Abstract
Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.
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Affiliation(s)
- Sang-Mi Kim
- Department of Biomedical Science, Graduate School, Hanyang University, Seoul 04763, Korea; Hanyang Biomedical Research Institute, Hanyang University, Seoul 04763, Korea
| | - Mi-Sun Lim
- R&D Center, Jeil Pharmaceutical Co., Ltd., Yongin 17172, Korea; Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul 06360, Korea
| | - Eun-Hye Lee
- Department of Biomedical Science, Graduate School, Hanyang University, Seoul 04763, Korea
| | - Sung Jun Jung
- Hanyang Biomedical Research Institute, Hanyang University, Seoul 04763, Korea; Department of Physiology, College of Medicine, Hanyang University, Seoul 04763, Korea
| | - Hee Yong Chung
- Hanyang Biomedical Research Institute, Hanyang University, Seoul 04763, Korea; Department of Microbiology, College of Medicine, Hanyang University, Seoul 04763, Korea; Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.
| | | | - Chang-Hwan Park
- Department of Biomedical Science, Graduate School, Hanyang University, Seoul 04763, Korea; Hanyang Biomedical Research Institute, Hanyang University, Seoul 04763, Korea; Department of Microbiology, College of Medicine, Hanyang University, Seoul 04763, Korea; Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.
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25
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Long J, Kim H, Kim D, Lee JB, Kim DH. A biomaterial approach to cell reprogramming and differentiation. J Mater Chem B 2017; 5:2375-2379. [PMID: 28966790 PMCID: PMC5616208 DOI: 10.1039/c6tb03130g] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Cell reprogramming of somatic cells into pluripotent states and subsequent differentiation into certain phenotypes has helped progress regenerative medicine research and other medical applications. Recent research has used viral vectors to induce this reprogramming; however, limitations include low efficiency and safety concerns. In this review, we discuss how biomaterial methods offer potential avenues for either increasing viability and downstream applicability of viral methods, or providing a safer alternative. The use of non-viral delivery systems, such as electroporation, micro/nanoparticles, nucleic acids and the modulation of culture substrate topography and stiffness have generated valuable insights regarding cell reprogramming.
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Affiliation(s)
- Joseph Long
- Department of Bioengineering, University of Washington, Seattle WA, 98195, USA
- Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine; University of Washington; Seattle, WA, 98109, USA
| | - Hyejin Kim
- Department of Chemical Engineering, University of Seoul, Seoul, 02504, South Korea
| | - Dajeong Kim
- Department of Chemical Engineering, University of Seoul, Seoul, 02504, South Korea
| | - Jong Bum Lee
- Department of Chemical Engineering, University of Seoul, Seoul, 02504, South Korea
| | - Deok-Ho Kim
- Department of Bioengineering, University of Washington, Seattle WA, 98195, USA
- Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine; University of Washington; Seattle, WA, 98109, USA
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26
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AKINCI E, YILDIZ M, ÜNAL P, BADAKUL G. In vitro transcription and validation of human pancreatic transcription factors’ mRNAs. Turk J Biol 2017. [DOI: 10.3906/biy-1610-29] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
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27
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Hepatocyte-like cells derived from induced pluripotent stem cells. Hepatol Int 2016; 11:54-69. [PMID: 27530815 DOI: 10.1007/s12072-016-9757-y] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Accepted: 07/19/2016] [Indexed: 12/24/2022]
Abstract
The discovery that coordinated expression of a limited number of genes can reprogram differentiated somatic cells to induced pluripotent stem cells (iPSC) has opened novel possibilities for developing cell-based models of diseases and regenerative medicine utilizing cell reprogramming or cell transplantation. Directed differentiation of iPSCs can potentially generate differentiated cells belonging to any germ layer, including cells with hepatocyte-like morphology and function. Such cells, termed iHeps, can be derived by sequential cell signaling using available information on embryological development or by forced expression of hepatocyte-enriched transcription factors. In addition to the translational aspects of iHeps, the experimental findings have provided insights into the mechanisms of cell plasticity that permit one cell type to transition to another. However, iHeps generated by current methods do not fully exhibit all characteristics of mature hepatocytes, highlighting the need for additional research in this area. Here we summarize the current approaches and achievements in this field and discuss some existing hurdles and emerging approaches for improving iPSC differentiation, as well as maintaining such cells in culture for increasing their utility in disease modeling and drug development.
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28
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Reautschnig P, Vogel P, Stafforst T. The notorious R.N.A. in the spotlight - drug or target for the treatment of disease. RNA Biol 2016; 14:651-668. [PMID: 27415589 PMCID: PMC5449091 DOI: 10.1080/15476286.2016.1208323] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
mRNA is an attractive drug target for therapeutic interventions. In this review we highlight the current state, clinical trials, and developments in antisense therapy, including the classical approaches like RNaseH-dependent oligomers, splice-switching oligomers, aptamers, and therapeutic RNA interference. Furthermore, we provide an overview on emerging concepts for using RNA in therapeutic settings including protein replacement by in-vitro-transcribed mRNAs, mRNA as vaccines and anti-allergic drugs. Finally, we give a brief outlook on early-stage RNA repair approaches that apply endogenous or engineered proteins in combination with short RNAs or chemically stabilized oligomers for the re-programming of point mutations, RNA modifications, and frame shift mutations directly on the endogenous mRNA.
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Affiliation(s)
- Philipp Reautschnig
- a Interfaculty Institute of Biochemistry, University of Tübingen Auf der Morgenstelle , Tübingen , Germany
| | - Paul Vogel
- a Interfaculty Institute of Biochemistry, University of Tübingen Auf der Morgenstelle , Tübingen , Germany
| | - Thorsten Stafforst
- a Interfaculty Institute of Biochemistry, University of Tübingen Auf der Morgenstelle , Tübingen , Germany
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29
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Steinle H, Behring A, Schlensak C, Wendel HP, Avci-Adali M. Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges. Stem Cells 2016; 35:68-79. [DOI: 10.1002/stem.2402] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2015] [Revised: 04/25/2016] [Accepted: 04/29/2016] [Indexed: 12/21/2022]
Affiliation(s)
- Heidrun Steinle
- Department of Thoracic and Cardiovascular Surgery; University Hospital Tuebingen; Calwerstraße 7/1 Tuebingen 72076 Germany
| | - Andreas Behring
- Department of Thoracic and Cardiovascular Surgery; University Hospital Tuebingen; Calwerstraße 7/1 Tuebingen 72076 Germany
| | - Christian Schlensak
- Department of Thoracic and Cardiovascular Surgery; University Hospital Tuebingen; Calwerstraße 7/1 Tuebingen 72076 Germany
| | - Hans Peter Wendel
- Department of Thoracic and Cardiovascular Surgery; University Hospital Tuebingen; Calwerstraße 7/1 Tuebingen 72076 Germany
| | - Meltem Avci-Adali
- Department of Thoracic and Cardiovascular Surgery; University Hospital Tuebingen; Calwerstraße 7/1 Tuebingen 72076 Germany
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30
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Rohani L, Fabian C, Holland H, Naaldijk Y, Dressel R, Löffler-Wirth H, Binder H, Arnold A, Stolzing A. Generation of human induced pluripotent stem cells using non-synthetic mRNA. Stem Cell Res 2016; 16:662-72. [DOI: 10.1016/j.scr.2016.03.008] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 02/28/2016] [Accepted: 03/17/2016] [Indexed: 11/24/2022] Open
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31
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The Importance of Ubiquitination and Deubiquitination in Cellular Reprogramming. Stem Cells Int 2016; 2016:6705927. [PMID: 26880980 PMCID: PMC4736574 DOI: 10.1155/2016/6705927] [Citation(s) in RCA: 76] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2015] [Revised: 11/04/2015] [Accepted: 11/12/2015] [Indexed: 01/09/2023] Open
Abstract
Ubiquitination of core stem cell transcription factors can directly affect stem cell maintenance and differentiation. Ubiquitination and deubiquitination must occur in a timely and well-coordinated manner to regulate the protein turnover of several stemness related proteins, resulting in optimal embryonic stem cell maintenance and differentiation. There are two switches: an E3 ubiquitin ligase enzyme that tags ubiquitin molecules to the target proteins for proteolysis and a second enzyme, the deubiquitinating enzyme (DUBs), that performs the opposite action, thereby preventing proteolysis. In order to maintain stemness and to allow for efficient differentiation, both ubiquitination and deubiquitination molecular switches must operate properly in a balanced manner. In this review, we have summarized the importance of the ubiquitination of core stem cell transcription factors, such as Oct3/4, c-Myc, Sox2, Klf4, Nanog, and LIN28, during cellular reprogramming. Furthermore, we emphasize the role of DUBs in regulating core stem cell transcriptional factors and their function in stem cell maintenance and differentiation. We also discuss the possibility of using DUBs, along with core transcription factors, to efficiently generate induced pluripotent stem cells. Our review provides a relatively new understanding regarding the importance of ubiquitination/deubiquitination of stem cell transcription factors for efficient cellular reprogramming.
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32
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iPSCs: A Minireview from Bench to Bed, including Organoids and the CRISPR System. Stem Cells Int 2016; 2016:5934782. [PMID: 26880972 PMCID: PMC4736429 DOI: 10.1155/2016/5934782] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2015] [Revised: 10/19/2015] [Accepted: 10/25/2015] [Indexed: 12/22/2022] Open
Abstract
When Dolly the sheep was born, the first probe into an adult mammalian genome traveling back in time and generating a whole new animal appeared. Ten years later, the reprogramming process became a defined method of producing induced pluripotent stem cells (iPSCs) through the overexpression of four transcription factors. iPSCs are capable of originating virtually all types of cells and tissues, including a whole new animal. The reprogramming strategies based on patient-derived cells should make the development of clinical applications of cell based therapy much more straightforward. Here, we analyze the current state, opportunities, and challenges of iPSCs from bench to bed, including organoids and the CRISPR system.
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33
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Messenger RNA delivery of a cartilage-anabolic transcription factor as a disease-modifying strategy for osteoarthritis treatment. Sci Rep 2016; 6:18743. [PMID: 26728350 PMCID: PMC4700530 DOI: 10.1038/srep18743] [Citation(s) in RCA: 87] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Accepted: 11/25/2015] [Indexed: 11/08/2022] Open
Abstract
Osteoarthritis (OA) is a chronic degenerative joint disease and a major health problem in the elderly population. No disease-modifying osteoarthritis drug (DMOAD) has been made available for clinical use. Here we present a disease-modifying strategy for OA, focusing on messenger RNA (mRNA) delivery of a therapeutic transcription factor using polyethylene glycol (PEG)-polyamino acid block copolymer-based polyplex nanomicelles. When polyplex nanomicelles carrying the cartilage-anabolic, runt-related transcription factor (RUNX) 1 mRNA were injected into mouse OA knee joints, OA progression was significantly suppressed compared with the non-treatment control. Expressions of cartilage-anabolic markers and proliferation were augmented in articular chondrocytes of the RUNX1-injected knees. Thus, this study provides a proof of concept of the treatment of degenerative diseases such as OA by the in situ mRNA delivery of therapeutic transcription factors; the presented approach will directly connect basic findings on disease-protective or tissue-regenerating factors to disease treatment.
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34
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Rhoads RE. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences. Methods Mol Biol 2016; 1428:3-27. [PMID: 27236789 DOI: 10.1007/978-1-4939-3625-0_1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA.
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Affiliation(s)
- Robert E Rhoads
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932, USA.
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Induced Pluripotency and Gene Editing in Disease Modelling: Perspectives and Challenges. Int J Mol Sci 2015; 16:28614-34. [PMID: 26633382 PMCID: PMC4691066 DOI: 10.3390/ijms161226119] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2015] [Revised: 11/13/2015] [Accepted: 11/24/2015] [Indexed: 02/07/2023] Open
Abstract
Embryonic stem cells (ESCs) are chiefly characterized by their ability to self-renew and to differentiate into any cell type derived from the three main germ layers. It was demonstrated that somatic cells could be reprogrammed to form induced pluripotent stem cells (iPSCs) via various strategies. Gene editing is a technique that can be used to make targeted changes in the genome, and the efficiency of this process has been significantly enhanced by recent advancements. The use of engineered endonucleases, such as homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Cas9 of the CRISPR system, has significantly enhanced the efficiency of gene editing. The combination of somatic cell reprogramming with gene editing enables us to model human diseases in vitro, in a manner considered superior to animal disease models. In this review, we discuss the various strategies of reprogramming and gene targeting with an emphasis on the current advancements and challenges of using these techniques to model human diseases.
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Poleganov MA, Eminli S, Beissert T, Herz S, Moon JI, Goldmann J, Beyer A, Heck R, Burkhart I, Barea Roldan D, Türeci Ö, Yi K, Hamilton B, Sahin U. Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion. Hum Gene Ther 2015; 26:751-66. [DOI: 10.1089/hum.2015.045] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Affiliation(s)
- Marco Alexander Poleganov
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany
| | | | - Tim Beissert
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Stephanie Herz
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- III. Department for Internal Medicine, Johannes Gutenberg University, Mainz, Germany
| | | | - Johanna Goldmann
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Arianne Beyer
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany
| | - Rosario Heck
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- III. Department for Internal Medicine, Johannes Gutenberg University, Mainz, Germany
| | - Isabell Burkhart
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Diana Barea Roldan
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- III. Department for Internal Medicine, Johannes Gutenberg University, Mainz, Germany
| | - Özlem Türeci
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Kevin Yi
- Stemgent, Cambridge, Massachusetts
| | | | - Ugur Sahin
- TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany
- III. Department for Internal Medicine, Johannes Gutenberg University, Mainz, Germany
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Preskey D, Allison TF, Jones M, Mamchaoui K, Unger C. Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts. Biochem Biophys Res Commun 2015; 473:743-51. [PMID: 26449459 DOI: 10.1016/j.bbrc.2015.09.102] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2015] [Accepted: 09/18/2015] [Indexed: 10/22/2022]
Abstract
Synthetic mRNA transfection enables efficient and controlled gene expression in human cells, without genome integrations. Further, modifications to the mRNA and transfection protocol now allow for repeated transfection and long-term gene expression of an otherwise short-lived mRNA expression. This is mainly achieved through introducing modified nucleosides and interferon suppression. In this study we provide an overview and details of the advanced synthesis and modifications of mRNA originally developed for reprogramming. This mRNA allows for very efficient transfection of fibroblasts enabling the generation of high quality human iPS cells with a six-factor mRNA cocktail in 9 days. Furthermore, we synthesised and transfected modified MYOD1 mRNA to transdifferentiate human fibroblasts into myoblast-like cells without a transgene footprint. This efficient and integration-free mRNA technology opens the door for safe and controlled gene expression to reverse or redirect cell fate.
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Affiliation(s)
- David Preskey
- Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK
| | - Thomas F Allison
- Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK
| | - Mark Jones
- Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK
| | - Kamel Mamchaoui
- Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, 47 Boulevard de l'hôpital, 75013 Paris, France
| | - Christian Unger
- Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK.
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Devoldere J, Dewitte H, De Smedt SC, Remaut K. Evading innate immunity in nonviral mRNA delivery: don't shoot the messenger. Drug Discov Today 2015. [PMID: 26210957 DOI: 10.1016/j.drudis.2015.07.009] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
In the field of nonviral gene therapy, in vitro transcribed (IVT) mRNA has emerged as a promising tool for the delivery of genetic information. Over the past few years it has become widely known that the introduction of IVT mRNA into mammalian cells elicits an innate immune response that has favored mRNA use toward immunotherapeutic vaccination strategies. However, for non-immunotherapy-related applications this intrinsic immune-stimulatory activity directly interferes with the aimed therapeutic outcome, because it can seriously compromise the expression of the desired protein. This review presents an overview of the immune-related obstacles that limit mRNA advance for non-immunotherapy-related applications.
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Affiliation(s)
- Joke Devoldere
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
| | - Heleen Dewitte
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
| | - Stefaan C De Smedt
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
| | - Katrien Remaut
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
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Guo YL, Carmichael GG, Wang R, Hong X, Acharya D, Huang F, Bai F. Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine. Stem Cells 2015; 33:3165-73. [PMID: 26086534 DOI: 10.1002/stem.2079] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2015] [Accepted: 05/16/2015] [Indexed: 12/14/2022]
Abstract
Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.
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Affiliation(s)
- Yan-Lin Guo
- The Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi, USA
| | - Gordon G Carmichael
- The Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA
| | - Ruoxing Wang
- The Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi, USA
| | - Xiaoxiao Hong
- The Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA
| | - Dhiraj Acharya
- The Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi, USA
| | - Faqing Huang
- The Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi, USA
| | - Fengwei Bai
- The Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi, USA
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Vallazza B, Petri S, Poleganov MA, Eberle F, Kuhn AN, Sahin U. Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond. WILEY INTERDISCIPLINARY REVIEWS-RNA 2015; 6:471-99. [DOI: 10.1002/wrna.1288] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/12/2015] [Revised: 04/23/2015] [Accepted: 04/28/2015] [Indexed: 12/24/2022]
Affiliation(s)
| | | | | | | | | | - Ugur Sahin
- BioNTech RNA Pharmaceuticals GmbH; Mainz Germany
- TRON gGmbH; Mainz Germany
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Kumar D, Talluri TR, Anand T, Kues WA. Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals. World J Stem Cells 2015; 7:315-328. [PMID: 25815117 PMCID: PMC4369489 DOI: 10.4252/wjsc.v7.i2.315] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 08/19/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.
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Revilla A, González C, Iriondo A, Fernández B, Prieto C, Marín C, Liste I. Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine. J Tissue Eng Regen Med 2015; 10:893-907. [PMID: 25758460 DOI: 10.1002/term.2021] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2014] [Revised: 12/19/2014] [Accepted: 01/26/2015] [Indexed: 12/20/2022]
Abstract
Over the last few years, the generation of induced pluripotent stem cells (iPSCs) from human somatic cells has proved to be one of the most potentially useful discoveries in regenerative medicine. iPSCs are becoming an invaluable tool to study the pathology of different diseases and for drug screening. However, several limitations still affect the possibility of applying iPS cell-based technology in therapeutic prospects. Most strategies for iPSCs generation are based on gene delivery via retroviral or lentiviral vectors, which integrate into the host's cell genome, causing a remarkable risk of insertional mutagenesis and oncogenic transformation. To avoid such risks, significant advances have been made with non-integrative reprogramming strategies. On the other hand, although many different kinds of somatic cells have been employed to generate iPSCs, there is still no consensus about the ideal type of cell to be reprogrammed. In this review we present the recent advances in the generation of human iPSCs, discussing their advantages and limitations in terms of safety and efficiency. We also present a selection of somatic cell sources, considering their capability to be reprogrammed and tissue accessibility. From a translational medicine perspective, these two topics will provide evidence to elucidate the most suitable combination of reprogramming strategy and cell source to be applied in each human iPSC-based therapy. The wide variety of diseases this technology could treat opens a hopeful future for regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.
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Affiliation(s)
- Ana Revilla
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Clara González
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Amaia Iriondo
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Bárbara Fernández
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Cristina Prieto
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Carlos Marín
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
| | - Isabel Liste
- Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain
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Pluripotent state induction in mouse embryonic fibroblast using mRNAs of reprogramming factors. Int J Mol Sci 2014; 15:21840-64. [PMID: 25437916 PMCID: PMC4284681 DOI: 10.3390/ijms151221840] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2014] [Revised: 10/31/2014] [Accepted: 11/05/2014] [Indexed: 12/12/2022] Open
Abstract
Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.
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Mehta A, Verma V, Nandihalli M, Ramachandra CJA, Sequiera GL, Sudibyo Y, Chung Y, Sun W, Shim W. A systemic evaluation of cardiac differentiation from mRNA reprogrammed human induced pluripotent stem cells. PLoS One 2014; 9:e103485. [PMID: 25068310 PMCID: PMC4113436 DOI: 10.1371/journal.pone.0103485] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 07/02/2014] [Indexed: 11/19/2022] Open
Abstract
Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by “foot-print free” reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.
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Affiliation(s)
- Ashish Mehta
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
- * E-mail: (AM); (WS)
| | - Vinod Verma
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
| | - Manasi Nandihalli
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
| | | | - Glen L. Sequiera
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
| | - Yuliansa Sudibyo
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
| | - Yingying Chung
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
| | - William Sun
- Experimental and Therapeutics Centre, A’STAR, Singapore, Singapore
| | - Winston Shim
- National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore
- Cardiovascular and Metabolic Disorders Program, DUKE-NUS, Singapore, Singapore
- * E-mail: (AM); (WS)
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Hu K. All roads lead to induced pluripotent stem cells: the technologies of iPSC generation. Stem Cells Dev 2014; 23:1285-300. [PMID: 24524728 PMCID: PMC4046204 DOI: 10.1089/scd.2013.0620] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Accepted: 02/12/2014] [Indexed: 12/26/2022] Open
Abstract
Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics, and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems, including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence, and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, nonintegrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, RNA virion, RNA replicon, nonintegrating replicating episomal plasmids, minicircles, polycistron, and preintegration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
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Affiliation(s)
- Kejin Hu
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Insitute, School of Medicine, University of Alabama at Birmingham , Birmingham, Alabama
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Van Pham P, Thi-My Nguyen P, Thai-Quynh Nguyen A, Minh Pham V, Nguyen-Tu Bui A, Thi-Tung Dang L, Gia Nguyen K, Kim Phan N. Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection. Differentiation 2014; 87:200-8. [DOI: 10.1016/j.diff.2014.08.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2014] [Revised: 08/04/2014] [Accepted: 08/18/2014] [Indexed: 02/08/2023]
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Ramakrishna S, Kim KS, Baek KH. Posttranslational modifications of defined embryonic reprogramming transcription factors. Cell Reprogram 2014; 16:108-20. [PMID: 24568610 DOI: 10.1089/cell.2013.0077] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The generation of induced pluripotent stem cells (iPSCs) from somatic cells by expressing ectopic reprogramming transcriptional factors such as Oct3/4, Sox2, Klf4, c-Myc, and Nanog is one of the cutting-edge discoveries in stem cell and cancer research. This discovery has raised several safety issues regarding the use of iPSC technology for human disease research. Tumorigenesis is the major obstacle observed for iPSC-mediated transplantation therapy. Recently, a new method to generate human iPSCs either by a chemical method or by direct delivery of reprogramming factors has become a promising approach for future customized cell therapy of human disorders. These reprogramming transcriptional factors play critical roles in diverse cellular functions such as transactivation, cellular proliferation, differentiation, apoptosis, and tumorigenesis. Posttranslational modifications (PTMs) (phosphorylation, ubiquitination, acetylation, sumoylation, and so on) of these proteins act as a regulatory signal to control protein activity, expression, and stability in a wide variety of cellular processes. We attempt to summarize the accumulated evidence to address the role of PTMs of Oct3/4, Sox2, Klf4, c-Myc, and Nanog in regulating their biological functions. This review allows us to understand the importance of PTMs and their application in developing an efficient and safe reprogramming method without cancer development for cell therapy. Finally, we discuss the importance of PTMs of reprogramming factors in tumor pathogenesis.
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Affiliation(s)
- Suresh Ramakrishna
- 1 Department of Biomedical Science, CHA University , Bundang CHA Hospital, Gyeonggi-Do, 463-840, Republic of Korea
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Sauer V, Roy-Chowdhury N, Guha C, Roy-Chowdhury J. Induced pluripotent stem cells as a source of hepatocytes. CURRENT PATHOBIOLOGY REPORTS 2014; 2:11-20. [PMID: 25650171 DOI: 10.1007/s40139-013-0039-2] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
During the past decade, a series of discoveries has established the potential of the so called terminally differentiated cells to transition to more primitive progenitor cells. The dramatic demonstration of the ability to reprogram differentiated somatic cells to induced pluripotent stem cells (iPSC) that can then give rise to cells of all three germ layers has opened the possibility of generating virtually any cell type in culture, from any given individual. Taking advantage of these concepts, researchers have generated iPSCs by reprogramming a wide variety of somatic cells. In addition to their practical implications, these studies have provided crucial insights into the mechanism of cell plasticity that underlies the transition from one cell type to another. Using concepts derived from research on embryological development, investigators have differentiated iPSCs to cells resembling hepatocytes in many ways. Such hepatocyte-like cells could be of enormous value in disease modeling, drug discovery and regenerative medicine. However, the currently available methods do not yield cells that fully reproduce the characteristics of adult primary hepatocytes. Thus generating hepatocytes from iPSCs is very much a work in progress. In addition to chronicling these exciting developments, this review will discuss the emergent new approaches to generating iPSCs, improving their differentiation to hepatocyte-like cells and maintaining the hepatocyte-like cells in culture for longer survival and better function.
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Affiliation(s)
- Vanessa Sauer
- Department of Medicine (Division of Gastroenterology and Hepatology), Albert Einstein College of Medicine, New York ; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York
| | - Namita Roy-Chowdhury
- Department of Medicine (Division of Gastroenterology and Hepatology), Albert Einstein College of Medicine, New York ; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York ; Department of Genetics, Albert Einstein College of Medicine, New York
| | - Chandan Guha
- Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York ; Department of Radiation Oncology, Albert Einstein College of Medicine, New York
| | - Jayanta Roy-Chowdhury
- Department of Medicine (Division of Gastroenterology and Hepatology), Albert Einstein College of Medicine, New York ; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York ; Department of Genetics, Albert Einstein College of Medicine, New York
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Wasik AM, Grabarek J, Pantovic A, Cieślar-Pobuda A, Asgari HR, Bundgaard-Nielsen C, Rafat M, Dixon IMC, Ghavami S, Łos MJ. Reprogramming and carcinogenesis--parallels and distinctions. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2014; 308:167-203. [PMID: 24411172 DOI: 10.1016/b978-0-12-800097-7.00005-1] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Rapid progress made in various areas of regenerative medicine in recent years occurred both at the cellular level, with the Nobel prize-winning discovery of reprogramming (generation of induced pluripotent stem (iPS) cells) and also at the biomaterial level. The use of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 (called commonly "Yamanaka factors") for the conversion of differentiated cells, back to the pluripotent/embryonic stage, has opened virtually endless and ethically acceptable source of stem cells for medical use. Various types of stem cells are becoming increasingly popular as starting components for the development of replacement tissues, or artificial organs. Interestingly, many of the transcription factors, key to the maintenance of stemness phenotype in various cells, are also overexpressed in cancer (stem) cells, and some of them may find the use as prognostic factors. In this review, we describe various methods of iPS creation, followed by overview of factors known to interfere with the efficiency of reprogramming. Next, we discuss similarities between cancer stem cells and various stem cell types. Final paragraphs are dedicated to interaction of biomaterials with tissues, various adverse reactions generated as a result of such interactions, and measures available, that allow for mitigation of such negative effects.
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Affiliation(s)
- Agata M Wasik
- Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - Jerzy Grabarek
- Department of Pathology, Pomeranian Medical University, Szczecin, Poland
| | - Aleksandar Pantovic
- Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, and Clinic of Neurology, Military Medical Academy, Belgrade, Serbia
| | - Artur Cieślar-Pobuda
- Department of Clinical and Experimental Medicine (IKE), Division of Cell Biology, and Integrative Regenerative Medicine Center (IGEN), Linköping University, Linköping, Sweden; Biosystems Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland
| | | | - Caspar Bundgaard-Nielsen
- Department of Clinical and Experimental Medicine (IKE), Division of Cell Biology, and Integrative Regenerative Medicine Center (IGEN), Linköping University, Linköping, Sweden; Laboratory for Stem Cell Research, Aalborg University, Aalborg, Denmark
| | - Mehrdad Rafat
- Department of Clinical and Experimental Medicine (IKE), Division of Cell Biology, and Integrative Regenerative Medicine Center (IGEN), Linköping University, Linköping, Sweden; Department of Biomedical Engineering (IMT), Linköping University, Linköping, Sweden
| | - Ian M C Dixon
- Department of Physiology, St. Boniface Research Centre, and Manitoba Institute of Child Health, University of Manitoba, Winnipeg, Canada
| | - Saeid Ghavami
- Department of Physiology, St. Boniface Research Centre, and Manitoba Institute of Child Health, University of Manitoba, Winnipeg, Canada
| | - Marek J Łos
- Department of Pathology, Pomeranian Medical University, Szczecin, Poland; Department of Clinical and Experimental Medicine (IKE), Division of Cell Biology, and Integrative Regenerative Medicine Center (IGEN), Linköping University, Linköping, Sweden; BioApplications Enterprises, Winnipeg, Manitoba, Canada.
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Wang R, Teng C, Spangler J, Wang J, Huang F, Guo YL. Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of mRNA-mediated gene expression strategy. Stem Cells Dev 2013; 23:594-604. [PMID: 24219369 DOI: 10.1089/scd.2013.0417] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine.
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Affiliation(s)
- Ruoxing Wang
- 1 Department of Biological Sciences, The University of Southern Mississippi , Hattiesburg, Mississippi
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