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Sung TC, Pan ZX, Wang T, Lin HY, Chang CL, Hung LC, Subbiah SK, Renuka RR, Chou SJ, Chiou SH, Joanna I, Lee HHC, Wu GJ, Higuchi A. Material surface conjugated with fibroblast growth factor-2 for pluripotent stem cell culture and differentiation. Regen Biomater 2025; 12:rbaf003. [PMID: 39967781 PMCID: PMC11835233 DOI: 10.1093/rb/rbaf003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2024] [Revised: 12/10/2024] [Accepted: 12/27/2024] [Indexed: 02/20/2025] Open
Abstract
Fibroblast growth factor-2 (FGF-2) is a critical molecule for sustaining the pluripotency of human pluripotent stem (PS) cells. However, FGF-2 is extremely unstable and cannot be stored long periods at room temperature. Therefore, the following FGF-2-conjugated cell culture materials were developed to stabilize FGF-2: FGF-2-conjugated polyvinyl alcohol (PVAI-C-FGF) hydrogels and FGF-2-conjugated carboxymethyl cellulose-coated (CMC-C-FGF) dishes. Human induced pluripotent stem (iPS) cells were proliferated on recombinant vitronectin (rVN)-coated PVAI-C-FGF hydrogels and CMC-C-FGF dishes in medium without FGF-2. Human iPS cells could not be cultivated on rVN-coated PVAI-C-FGF hydrogels for more than two passages but could proliferate on rVN-coated CMC-C-FGF dishes. These results indicated that the amount of immobilized FGF-2 and the base cell materials are important, including the amount of immobilized rVN and the conformation of FGF-2 on the surfaces. When human iPS cells were proliferated on rVN-coated CMC-C-FGF surfaces in medium containing no FGF-2 for 10 passages, their pluripotency and potential to differentiate into cells originating from three germ layers were maintained in vivo and in vitro. Furthermore, the cells could extensively differentiate into cardiomyocytes, which can be used for cardiac infarction treatment in future and retinal pigment epithelium for retinal pigmentosa treatment in future. The FGF-2-immobilized surface could enable human PS cell culture in medium that does not need to contain unstable FGF-2. The amount of FGF-2 immobilization on the rVN-coated CMC-C-5FGF and CMC-C-20FGF dishes was reduced to 93.6 and 52.2 times, respectively, which is less than the conventional amount of FGF-2 used in culture medium for one passage (6 days) of human iPS cell culture. This reduction resulted from the stabilization of unstable FGF-2 by the immobilization of FGF-2, which was achieved by utilizing optimal base materials (CMC), coating materials (rVN) and long-joint segment (PEG4-SPDP) design.
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Affiliation(s)
- Tzu-Cheng Sung
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
| | - Zhi-Xian Pan
- Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan, ROC
| | - Ting Wang
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
| | - Hui-Yu Lin
- Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan, ROC
| | - Chia-Lun Chang
- Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan, ROC
| | - Ling-Chun Hung
- Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan, ROC
| | - Suresh Kumar Subbiah
- Centre for Stem Cell Mediated Advanced Research Therapeutics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 600 077, Tamil Nadu, India
| | - Remya Rajan Renuka
- Centre for Stem Cell Mediated Advanced Research Therapeutics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 600 077, Tamil Nadu, India
| | - Shih-Jie Chou
- Department of Medical Research, Taipei Veterans General Hospital, Taipei 112201, Taiwan, ROC
- Institute of Pharmacology, School of Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan, ROC
| | - Shih-Hwa Chiou
- Department of Medical Research, Taipei Veterans General Hospital, Taipei 112201, Taiwan, ROC
- Institute of Pharmacology, School of Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan, ROC
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei 11217, Taiwan, ROC
| | - Idaszek Joanna
- Division of Materials Design, Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw 02-507, Poland
| | - Henry Hsin-Chung Lee
- Department of Surgery, Hsinchu Cathay General Hospital, Hsinchu 30060, Taiwan, ROC
- Graduate Institute of Translational and Interdisciplinary Medicine, National Central University, Taoyuan 32001, Taiwan, ROC
| | - Gwo-Jang Wu
- Graduate Institute of Medical Sciences and Department of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan, ROC
| | - Akon Higuchi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
- Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan, ROC
- R&D Center for Membrane Technology, Chung Yuan Christian University, Taoyuan 320, Taiwan, ROC
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Kalia AK, Rösseler C, Granja-Vazquez R, Ahmad A, Pancrazio JJ, Neureiter A, Zhang M, Sauter D, Vetter I, Andersson A, Dussor G, Price TJ, Kolber BJ, Truong V, Walsh P, Lampert A. How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment. Stem Cell Res Ther 2024; 15:99. [PMID: 38581069 PMCID: PMC10998320 DOI: 10.1186/s13287-024-03696-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Accepted: 03/13/2024] [Indexed: 04/07/2024] Open
Abstract
BACKGROUND Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.
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Affiliation(s)
- Anil Kumar Kalia
- Institute of Neurophysiology, Uniklinik RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany
- Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, Aachen, Germany
| | - Corinna Rösseler
- Institute of Neurophysiology, Uniklinik RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany
| | - Rafael Granja-Vazquez
- Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, 75080, USA
- Department of Bioengineering, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Ayesha Ahmad
- Department of Neuroscience, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Joseph J Pancrazio
- Department of Bioengineering, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Anika Neureiter
- Institute of Neurophysiology, Uniklinik RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany
| | - Mei Zhang
- Sophion Bioscience Inc., Bedford, MA, 01730, USA
| | | | - Irina Vetter
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia
| | - Asa Andersson
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia
| | - Gregory Dussor
- Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, 75080, USA
- Department of Neuroscience, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Theodore J Price
- Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, 75080, USA
- Department of Neuroscience, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Benedict J Kolber
- Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, 75080, USA
- Department of Neuroscience, University of Texas at Dallas, Richardson, TX, 75080, USA
| | - Vincent Truong
- Anatomic Incorporated, 2112 Broadway Street NE #135, Minneapolis, MN, 55413, USA
| | - Patrick Walsh
- Anatomic Incorporated, 2112 Broadway Street NE #135, Minneapolis, MN, 55413, USA
| | - Angelika Lampert
- Institute of Neurophysiology, Uniklinik RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
- Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, Aachen, Germany.
- Scientific Center for Neuropathic Pain Aachen - SCN-Aachen, Uniklinik RWTH Aachen University, 52074, Aachen, Germany.
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Gallego Villarejo L, Gerding WM, Bachmann L, Hardt LHI, Bormann S, Nguyen HP, Müller T. Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research. Cells 2024; 13:507. [PMID: 38534351 PMCID: PMC10969360 DOI: 10.3390/cells13060507] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Revised: 03/02/2024] [Accepted: 03/05/2024] [Indexed: 03/28/2024] Open
Abstract
Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.
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Affiliation(s)
- Lucia Gallego Villarejo
- Department of Molecular Biochemistry, Ruhr-University Bochum, 44801 Bochum, Germany; (L.B.); (L.H.I.H.); (S.B.)
- Department of Cytology, Institute of Anatomy, Ruhr-University Bochum, 44801 Bochum, Germany
- International Graduate School of Neuroscience, Ruhr-University Bochum, 44801 Bochum, Germany
| | - Wanda M. Gerding
- Department of Human Genetics, Ruhr-University Bochum, 44801 Bochum, Germany; (W.M.G.); (H.P.N.)
| | - Lisa Bachmann
- Department of Molecular Biochemistry, Ruhr-University Bochum, 44801 Bochum, Germany; (L.B.); (L.H.I.H.); (S.B.)
| | - Luzie H. I. Hardt
- Department of Molecular Biochemistry, Ruhr-University Bochum, 44801 Bochum, Germany; (L.B.); (L.H.I.H.); (S.B.)
| | - Stefan Bormann
- Department of Molecular Biochemistry, Ruhr-University Bochum, 44801 Bochum, Germany; (L.B.); (L.H.I.H.); (S.B.)
| | - Huu Phuc Nguyen
- Department of Human Genetics, Ruhr-University Bochum, 44801 Bochum, Germany; (W.M.G.); (H.P.N.)
| | - Thorsten Müller
- Institute of Psychiatric Phenomics and Genomics (IPPG), University Hospital, LMU Munich, 80336 Munich, Germany;
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Kalia AK, Rösseler C, Granja-Vazquez R, Ahmad A, Pancrazio JJ, Neureiter A, Zhang M, Sauter D, Vetter I, Andersson A, Dussor G, Price TJ, Kolber BJ, Truong V, Walsh P, Lampert A. How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols. RESEARCH SQUARE 2023:rs.3.rs-3127017. [PMID: 37961300 PMCID: PMC10635298 DOI: 10.21203/rs.3.rs-3127017/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2023]
Abstract
Background Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic symptoms. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs for disease modelling remain key challenges to study human nociception in vitro. Here, we report a detailed characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Methods Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Expression profiling of sensory neurons was performed with Immunocytochemistry and in situ hybridization techniques. Manual patch clamp and high throughput cellular screening systems (Fluorescence imaging plate reader, automated patch clamp and multi-well microelectrode arrays recordings) were applied to functionally characterize the generated sensory neurons. Results The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. High throughput systems confirmed functional expression of Na+ and K+ ion channels. Multi-well microelectrode recordings display spontaneously active neurons with sensitivity to increased temperature indicating expression of heat sensitive ion channels. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. Conclusions We validated the efficiency of two differentiation protocols and their potential application for understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.
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Affiliation(s)
| | | | | | | | | | | | - Mei Zhang
- Sophion Bioscience A/S: Biolin Scientific AB
| | | | - Irina Vetter
- The University of Queensland Institute for Molecular Bioscience
| | - Asa Andersson
- The University of Queensland Institute for Molecular Bioscience
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Lam ATL, Ho V, Vassilev S, Reuveny S, Oh SKW. An allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers. Cell Prolif 2022; 55:e13256. [PMID: 36574589 PMCID: PMC9357361 DOI: 10.1111/cpr.13256] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 04/19/2022] [Accepted: 04/28/2022] [Indexed: 12/30/2022] Open
Abstract
OBJECTIVES Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. MATERIALS AND METHODS Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. RESULTS Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. CONCLUSIONS Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.
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Affiliation(s)
- Alan Tin Lun Lam
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Valerie Ho
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Svetlan Vassilev
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Shaul Reuveny
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Steve Kah Weng Oh
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
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Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells. Mol Biol Rep 2022; 49:7887-7898. [PMID: 35637316 DOI: 10.1007/s11033-022-07621-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 05/19/2022] [Indexed: 01/31/2023]
Abstract
BACKGROUND Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.
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Potential and challenges of placenta-derived decidua stromal cell therapy in inflammation-associated disorders. Hum Immunol 2022; 83:580-588. [DOI: 10.1016/j.humimm.2022.04.011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2022] [Revised: 04/07/2022] [Accepted: 04/24/2022] [Indexed: 01/08/2023]
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Picerno A, Stasi A, Franzin R, Curci C, di Bari I, Gesualdo L, Sallustio F. Why stem/progenitor cells lose their regenerative potential. World J Stem Cells 2021; 13:1714-1732. [PMID: 34909119 PMCID: PMC8641024 DOI: 10.4252/wjsc.v13.i11.1714] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 05/26/2021] [Accepted: 10/31/2021] [Indexed: 02/06/2023] Open
Abstract
Nowadays, it is clear that adult stem cells, also called as tissue stem cells, play a central role to repair and maintain the tissue in which they reside by their self-renewal ability and capacity of differentiating into distinct and specialized cells. As stem cells age, their renewal ability declines and their capacity to maintain organ homeostasis and regeneration is impaired. From a molecular perspective, these changes in stem cells properties can be due to several types of cell intrinsic injury and DNA aberrant alteration (i.e epigenomic profile) as well as changes in the tissue microenviroment, both into the niche and by systemic circulating factors. Strikingly, it has been suggested that aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Therefore, understanding how resident stem cell age and affects near and distant tissues is fundamental. Here, we examine the current knowledge about aging mechanisms in several kinds of adult stem cells under physiological and pathological conditions and the principal aging-related changes in number, function and phenotype that determine the loss of tissue renewal properties. Furthermore, we examine the possible cell rejuvenation strategies. Stem cell rejuvenation may reverse the aging phenotype and the discovery of effective methods for inducing and differentiating pluripotent stem cells for cell replacement therapies could open up new possibilities for treating age-related diseases.
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Affiliation(s)
- Angela Picerno
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Alessandra Stasi
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Rossana Franzin
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Claudia Curci
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Ighli di Bari
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Loreto Gesualdo
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Fabio Sallustio
- Department of Interdisciplinary Medicine, University of Bari "Aldo Moro", Bari 70124, Italy
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Kim MH, Thanuthanakhun N, Fujimoto S, Kino-Oka M. Effect of initial seeding density on cell behavior-driven epigenetic memory and preferential lineage differentiation of human iPSCs. Stem Cell Res 2021; 56:102534. [PMID: 34530397 DOI: 10.1016/j.scr.2021.102534] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2021] [Revised: 08/22/2021] [Accepted: 09/03/2021] [Indexed: 10/20/2022] Open
Abstract
Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent state and subsequent differentiation. Interestingly, pluripotency-associated genes were activated during the entire cell growth phases along with increased active modifications and decreased repressive modifications. This memory effect can last several days in the long-term stationary phase and was sustained in the aspect of cell behavioral changes after subculture. Further, changes in growth-related cell behavior were found to induce nucleoskeletal reorganization and active versus repressive modifications, thereby enabling hiPSCs to change their differentiation potential. Overall, we discuss the cell behavior-driven epigenetic memory induced by the culture environment, and the effect of previous memory on cell lineage specification in the process of hiPSC differentiation.
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Affiliation(s)
- Mee-Hae Kim
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
| | - Naruchit Thanuthanakhun
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Shun Fujimoto
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Masahiro Kino-Oka
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
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10
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Skorik C, Mullin NK, Shi M, Zhang Y, Hunter P, Tang Y, Hilton B, Schlaeger TM. Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521. ACTA ACUST UNITED AC 2021; 52:e103. [PMID: 31977148 DOI: 10.1002/cpsc.103] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component-free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent-derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2: PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2: Testing Sendai virus kit-reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3: Testing Epi5 kit-reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion.
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Affiliation(s)
- Christian Skorik
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Stemcell Technologies, Cambridge, Massachusetts
| | - Nathaniel K Mullin
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Michael Shi
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,School of Medicine, Case Western Reserve University, Cleveland, Ohio
| | - Yosra Zhang
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Stemcell Technologies, Cambridge, Massachusetts
| | - Phoebe Hunter
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Yang Tang
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Brianna Hilton
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Thorsten M Schlaeger
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Harvard Stem Cell Institute, Boston, Massachusetts.,Harvard Medical School, Boston, Massachusetts
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11
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Wang Y, Sims CE, Allbritton NL. Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells. Anal Chem 2020; 92:12330-12337. [PMID: 32819098 DOI: 10.1021/acs.analchem.0c02016] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.
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Affiliation(s)
- Yuli Wang
- Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States.,Department of Bioengineering, University of Washington, Seattle, Washington 98105, United States
| | - Christopher E Sims
- Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States.,Department of Bioengineering, University of Washington, Seattle, Washington 98105, United States
| | - Nancy L Allbritton
- Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States.,Department of Bioengineering, University of Washington, Seattle, Washington 98105, United States
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12
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Paterson YZ, Cribbs A, Espenel M, Smith EJ, Henson FMD, Guest DJ. Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes. Stem Cell Res Ther 2020; 11:184. [PMID: 32430075 PMCID: PMC7238619 DOI: 10.1186/s13287-020-01692-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 04/21/2020] [Accepted: 04/28/2020] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic. METHODS Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing. RESULTS No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes' transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells' gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed. CONCLUSION The information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.
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Affiliation(s)
- Y. Z. Paterson
- Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES UK
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - A. Cribbs
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7LD UK
| | - M. Espenel
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - E. J. Smith
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - F. M. D. Henson
- Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES UK
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - D. J. Guest
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
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13
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Steichen C, Hannoun Z, Luce E, Hauet T, Dubart-Kupperschmitt A. Genomic integrity of human induced pluripotent stem cells: Reprogramming, differentiation and applications. World J Stem Cells 2019; 11:729-747. [PMID: 31692979 PMCID: PMC6828592 DOI: 10.4252/wjsc.v11.i10.729] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Revised: 06/13/2019] [Accepted: 07/30/2019] [Indexed: 02/06/2023] Open
Abstract
Ten years after the initial generation of induced pluripotent stem cells (hiPSCs) from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest. Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.
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Affiliation(s)
- Clara Steichen
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers F-86021, France
- Université de Poitiers, Faculté de Médecine et Pharmacie, Bâtiment D1, 6 rue de la milétrie, TSA 51115, 86073 Poitiers Cedex 9, France
| | - Zara Hannoun
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- The Jenner Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom
| | - Eléanor Luce
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- Département Hospitalo-Universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Thierry Hauet
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers F-86021, France
- Université de Poitiers, Faculté de Médecine et Pharmacie, Bâtiment D1, 6 rue de la milétrie, TSA 51115, 86073 Poitiers Cedex 9, France
- Service de Biochimie, Pôle Biospharm, CHU de Poitiers, Poitiers F-86021, France
- Fédération Hospitalo-Universitaire SUPORT, CHU de Poitiers, Poitiers F-86021, France
| | - Anne Dubart-Kupperschmitt
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- Département Hospitalo-Universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
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14
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Hu J, Wang J. From embryonic stem cells to induced pluripotent stem cells-Ready for clinical therapy? Clin Transplant 2019; 33:e13573. [PMID: 31013374 DOI: 10.1111/ctr.13573] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 04/18/2019] [Indexed: 01/08/2023]
Abstract
Embryonic stem cells and induced pluripotent stem cells have increasingly important roles in many different fields of research and medicine. Major areas of impact include improved in vitro disease models, drug screening, and the development of cell-based clinical therapies. Here, we review the generation and uses of embryonic stem cells compared to induced pluripotent stem cells and discuss their advantages and limitations. We also evaluate the feasibility of clinical therapies and the future prospects for induced pluripotent cell-based treatments.
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Affiliation(s)
- Jing Hu
- Department of Neonatology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Jimei Wang
- Department of Neonatology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
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15
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Singh AM. An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells. Front Cell Dev Biol 2019; 7:11. [PMID: 30766873 PMCID: PMC6365467 DOI: 10.3389/fcell.2019.00011] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2018] [Accepted: 01/18/2019] [Indexed: 01/09/2023] Open
Abstract
Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7–10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1–2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.
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Affiliation(s)
- Amar M Singh
- Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, University of Georgia, Athens, GA, United States
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16
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Aramburú Junior JS, Eilers Treichel TL, Lemos Pinto Filho ST, Gehrke SA, Machado AK, Cadoná FC, Mânica da Cruz IB, Pippi NL. DNA damage in dental pulp mesenchymal stem cells: An in vitro study. VETERINARY RESEARCH FORUM : AN INTERNATIONAL QUARTERLY JOURNAL 2018; 9:293-299. [PMID: 30713606 PMCID: PMC6346493 DOI: 10.30466/vrf.2018.33083] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 11/07/2017] [Indexed: 12/21/2022]
Abstract
The aim of this study was to evaluate the potential use of a DNA comet assay, DNA fragmentation fluorimetric assay and reactive oxygen species levels as potential biomarkers of genome conditions of dental pulp stem cells (DPSCs) isolated from dog canine teeth. Mesenchymal stem cells were isolated from the dental pulp collected from dog teeth. The results obtained suggest the ideal moment for clinical application of cellular therapy for this type of cell. The cell culture was maintained with Dulbecco’s modified Eagle’s medium supplemented with 10.00% fetal bovine serum for eight passages. During each passage, cell proliferation, oxidative stress and level of DNA fragmentation were assessed by3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium (MTT) assay, testing 2,7 dichlorodihydro-fluorescein-diacetate and PicoGreen®, respectively. There were important differences among the first three DPSC passages compared to passages 4–8 and a large number of nuclei with some levels of DNA damage (30.00 to 40.00% in initial DPSC passages and > 50.00% in late passages), indicating in vitro DPSC genomic fragility. Within the limitations of this study, the results suggest these relatively simple and inexpensive approaches - comet and DNA fragmentation assays - could help sort stem cells with less DNA damage for use in research or therapies.
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Affiliation(s)
- Jaime Sardá Aramburú Junior
- Graduate Program in Veterinary Medicine, Federal University of Santa Maria, Santa Maria, Brazil.,Biotecnos Research Center, Santa Maria, Rio Grande do Sul, Brazil; Catholic University of Uruguay, Montevideo, Uruguay
| | | | | | - Sergio Alexandre Gehrke
- Biotecnos Research Center, Santa Maria, Rio Grande do Sul, Brazil; Catholic University of Uruguay, Montevideo, Uruguay
| | | | | | | | - Ney Luis Pippi
- Graduate Program in Veterinary Medicine, Federal University of Santa Maria, Santa Maria, Brazil
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17
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Chen YH, Pruett-Miller SM. Improving single-cell cloning workflow for gene editing in human pluripotent stem cells. Stem Cell Res 2018; 31:186-192. [PMID: 30099335 DOI: 10.1016/j.scr.2018.08.003] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Revised: 07/27/2018] [Accepted: 08/02/2018] [Indexed: 01/01/2023] Open
Abstract
The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting. Stem-Flex and RevitaCell supplement, in combination with Geltrex or Vitronectin (VN), promote reliable single-cell growth in a feeder-free and defined environment. Characterization of final genome-edited clones reveals that pluripotency and normal karyotype are retained following this single-cell culture protocol. This time-efficient and simplified culture method paves the way for high-throughput hPSC culture and will be valuable for both basic research and clinical applications.
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Affiliation(s)
- Yi-Hsien Chen
- Washington University School of Medicine, Department of Genetics, St. Louis 63110, USA; Genome Engineering and iPSC Center, USA.
| | - Shondra M Pruett-Miller
- St. Jude Children's Research Hospital, Department of Cell & Molecular Biology, Memphis 38105, USA; Center for Advanced Genome Engineering, USA.
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18
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Alimohamed MZ, Johansson LF, de Boer EN, Splinter E, Klous P, Yilmaz M, Bosga A, van Min M, Mulder AB, Vellenga E, Sinke RJ, Sijmons RH, van den Berg E, Sikkema-Raddatz B. Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification. Clin Chem 2018; 64:1096-1103. [DOI: 10.1373/clinchem.2017.286047] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Accepted: 04/16/2018] [Indexed: 11/06/2022]
Abstract
Abstract
BACKGROUND
Over 500 translocations have been identified in acute leukemia. To detect them, most diagnostic laboratories use karyotyping, fluorescent in situ hybridization, and reverse transcription PCR. Targeted locus amplification (TLA), a technique using next-generation sequencing, now allows detection of the translocation partner of a specific gene, regardless of its chromosomal origin. We present a TLA multiplex assay as a potential first-tier screening test for detecting translocations in leukemia diagnostics.
METHODS
The panel includes 17 genes involved in many translocations present in acute leukemias. Procedures were optimized by using a training set of cell line dilutions and 17 leukemia patient bone marrow samples and validated by using a test set of cell line dilutions and a further 19 patient bone marrow samples. Per gene, we determined if its region was involved in a translocation and, if so, the translocation partner. To balance sensitivity and specificity, we introduced a gray zone showing indeterminate translocation calls needing confirmation. We benchmarked our method against results from the 3 standard diagnostic tests.
RESULTS
In patient samples passing QC, we achieved a concordance with benchmarking tests of 81% in the training set and 100% in the test set, after confirmation of 4 and nullification of 3 gray zone calls (in total). In cell line dilutions, we detected translocations in 10% aberrant cells at several genetic loci.
CONCLUSIONS
Multiplex TLA shows promising results as an acute leukemia screening test. It can detect cryptic and other translocations in selected genes. Further optimization may make this assay suitable for diagnostic use.
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Affiliation(s)
- Mohamed Z Alimohamed
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | - Lennart F Johansson
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
- University of Groningen, University Medical Center Groningen, Genomics Coordination Center, Groningen, the Netherlands
| | - Eddy N de Boer
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | | | | | | | - Anneke Bosga
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | | | - André B Mulder
- University of Groningen, University Medical Center Groningen, Department of Laboratory Medicine, the Netherlands
| | - Edo Vellenga
- University of Groningen, University Medical Center Groningen, Department of Hematology, the Netherlands
| | - Richard J Sinke
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | - Rolf H Sijmons
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | - Eva van den Berg
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
| | - Birgit Sikkema-Raddatz
- University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands
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19
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Assou S, Bouckenheimer J, De Vos J. Concise Review: Assessing the Genome Integrity of Human Induced Pluripotent Stem Cells: What Quality Control Metrics? Stem Cells 2018; 36:814-821. [PMID: 29441649 DOI: 10.1002/stem.2797] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 01/19/2018] [Accepted: 01/24/2018] [Indexed: 12/20/2022]
Abstract
Human induced pluripotent stem cells (hiPSCs) have the potential to differentiate virtually into any cell type in unlimited quantities. Therefore, they are ideal for in vitro tissue modeling or to produce cells for clinical use. Importantly, and differently from immortalized and cancer cell lines, the hiPSC genome scrupulously reproduces that of the cell from which they were derived. However, hiPSCs can develop genetic abnormalities during reprogramming or prolonged cell culture, such as aneuploidies or oncogenic mutations (e.g., in TP53). Therefore, hiPSC genome integrity must be routinely monitored because serious genome alterations would greatly compromise their usefulness or safety of use. Here, we reviewed hiPSC genome quality control monitoring methods and laboratory practice. Indeed, due to their frequency and functional consequences, recurrent genetic defects found in cultured hiPSCs are inacceptable and their appearance should be monitored by routine screening. Hence, for research purposes, we propose that the genome of hiPSC lines should be systematically screened at derivation, at least by karyotyping, and then regularly (every 12 weeks) during experiments, for instance with polymerase chain reaction-based techniques. For some specific applications, such as research on aging, cell cycle, apoptosis or cancer, other tests (e.g., TP53 mutation detection) should also be included. For clinical use, in addition to karyotyping, we advise exome sequencing. Stem Cells 2018;36:814-821.
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Affiliation(s)
- Said Assou
- IRMB, Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France
| | | | - John De Vos
- IRMB, Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France.,CHU Montpellier, Department of Cell and Tissue Engineering, Hospital Saint-Eloi, Montpellier, France
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20
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Chen X, Harkness L, Jia Z, Prowse A, Monteiro MJ, Gray PP. Methods for Expansion of Three-Dimensional Cultures of Human Embryonic Stem Cells Using a Thermoresponsive Polymer. Tissue Eng Part C Methods 2017; 24:146-157. [PMID: 29239281 DOI: 10.1089/ten.tec.2017.0331] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.
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Affiliation(s)
- Xiaoli Chen
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Linda Harkness
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Zhongfan Jia
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Andrew Prowse
- 2 The Garvan Institute of Medical Research , Sydney, Australia
| | - Michael J Monteiro
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Peter P Gray
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
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21
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Martin U. Genome stability of programmed stem cell products. Adv Drug Deliv Rev 2017; 120:108-117. [PMID: 28917518 DOI: 10.1016/j.addr.2017.09.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Revised: 08/31/2017] [Accepted: 09/07/2017] [Indexed: 01/23/2023]
Abstract
Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion.
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Affiliation(s)
- Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH Cluster of Excellence, German Center for Lung Research, Hannover Medical School, Germany.
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22
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Skamagki M, Correia C, Yeung P, Baslan T, Beck S, Zhang C, Ross CA, Dang L, Liu Z, Giunta S, Chang TP, Wang J, Ananthanarayanan A, Bohndorf M, Bosbach B, Adjaye J, Funabiki H, Kim J, Lowe S, Collins JJ, Lu CW, Li H, Zhao R, Kim K. ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors. Nat Cell Biol 2017; 19:1037-1048. [PMID: 28846095 PMCID: PMC5843481 DOI: 10.1038/ncb3598] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Accepted: 07/24/2017] [Indexed: 02/06/2023]
Abstract
Induced pluripotent stem cells (iPSCs), which are used to produce transplantable tissues, may particularly benefit older patients, who are more likely to suffer from degenerative diseases. However, iPSCs generated from aged donors (A-iPSCs) exhibit higher genomic instability, defects in apoptosis and a blunted DNA damage response compared with iPSCs generated from younger donors. We demonstrated that A-iPSCs exhibit excessive glutathione-mediated reactive oxygen species (ROS) scavenging activity, which blocks the DNA damage response and apoptosis and permits survival of cells with genomic instability. We found that the pluripotency factor ZSCAN10 is poorly expressed in A-iPSCs and addition of ZSCAN10 to the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) during A-iPSC reprogramming normalizes ROS-glutathione homeostasis and the DNA damage response, and recovers genomic stability. Correcting the genomic instability of A-iPSCs will ultimately enhance our ability to produce histocompatible functional tissues from older patients' own cells that are safe for transplantation.
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Affiliation(s)
- Maria Skamagki
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
| | - Cristina Correia
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55904, USA
| | - Percy Yeung
- Department of Obstetrics, Gynecology and Reproductive Sciences, Child Health Institute of New Jersey, New Brunswick, New Jersey 08901, USA
| | - Timour Baslan
- Howard Hughes Medical Institute, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA
| | - Samuel Beck
- Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
| | - Cheng Zhang
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55904, USA
| | - Christian A. Ross
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55904, USA
| | - Lam Dang
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
| | - Zhong Liu
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
| | - Simona Giunta
- Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, New York 10065, USA
| | - Tzu-Pei Chang
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
| | - Joye Wang
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
| | - Aparna Ananthanarayanan
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
| | - Martina Bohndorf
- Institute for Stem Cell Research and Regenerative Medicine, Heinrich Heine University, Düsseldorf D-40225, Germany
| | - Benedikt Bosbach
- Howard Hughes Medical Institute, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA
| | - James Adjaye
- Institute for Stem Cell Research and Regenerative Medicine, Heinrich Heine University, Düsseldorf D-40225, Germany
| | - Hironori Funabiki
- Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, New York 10065, USA
| | - Jonghwan Kim
- Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
| | - Scott Lowe
- Howard Hughes Medical Institute, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA
| | - James J. Collins
- Department of Biological Engineering, Massachusetts Institute of Technology, Broad Institute of MIT and Harvard, and Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02118, USA
| | - Chi-Wei Lu
- Department of Obstetrics, Gynecology and Reproductive Sciences, Child Health Institute of New Jersey, New Brunswick, New Jersey 08901, USA
| | - Hu Li
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55904, USA
| | - Rui Zhao
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
| | - Kitai Kim
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute for Cancer Research, and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA
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Gu Q, Tomaskovic‐Crook E, Wallace GG, Crook JM. 3D Bioprinting Human Induced Pluripotent Stem Cell Constructs for In Situ Cell Proliferation and Successive Multilineage Differentiation. Adv Healthc Mater 2017; 6. [PMID: 28544655 DOI: 10.1002/adhm.201700175] [Citation(s) in RCA: 125] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Revised: 04/25/2017] [Indexed: 01/01/2023]
Abstract
The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine, including individualized, patient-specific stem cell-based treatments. There are, however, few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues, ideally comprising direct-write printing of cells for encapsulation, proliferation, and differentiation. Here, such a method, employing a clinically amenable polysaccharide-based bioink, is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically, we have extrusion printed the bioink including iPSCs, alginate (Al; 5% weight/volume [w/v]), carboxymethyl-chitosan (5% w/v), and agarose (Ag; 1.5% w/v), crosslinked the bioink in calcium chloride for a stable and porous construct, proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm, ectoderm, and mesoderm, or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined, scalable, and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine.
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Affiliation(s)
- Qi Gu
- ARC Centre of Excellence for Electromaterials Science Intelligent Polymer Research Institute AIIM Facility Innovation Campus University of Wollongong Squires Way Fairy Meadow New South Wales 2519 Australia
- State Key Laboratory of Stem Cell and Reproductive Biology Institute of Zoology Chinese Academy of Sciences Beijing 100101 P. R. China
| | - Eva Tomaskovic‐Crook
- ARC Centre of Excellence for Electromaterials Science Intelligent Polymer Research Institute AIIM Facility Innovation Campus University of Wollongong Squires Way Fairy Meadow New South Wales 2519 Australia
- Illawarra Health and Medical Research Institute University of Wollongong Wollongong New South Wales 2522 Australia
| | - Gordon G. Wallace
- ARC Centre of Excellence for Electromaterials Science Intelligent Polymer Research Institute AIIM Facility Innovation Campus University of Wollongong Squires Way Fairy Meadow New South Wales 2519 Australia
| | - Jeremy M. Crook
- ARC Centre of Excellence for Electromaterials Science Intelligent Polymer Research Institute AIIM Facility Innovation Campus University of Wollongong Squires Way Fairy Meadow New South Wales 2519 Australia
- Illawarra Health and Medical Research Institute University of Wollongong Wollongong New South Wales 2522 Australia
- Department of Surgery St Vincent's Hospital The University of Melbourne Fitzroy Victoria 3065 Australia
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24
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Kovac M, Vasicek J, Kulikova B, Bauer M, Curlej J, Balazi A, Chrenek P. Different RNA and protein expression of surface markers in rabbit amniotic fluid-derived mesenchymal stem cells. Biotechnol Prog 2017; 33:1601-1613. [DOI: 10.1002/btpr.2519] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2017] [Revised: 05/25/2017] [Indexed: 12/12/2022]
Affiliation(s)
- Michal Kovac
- Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture; Nitra Slovak Republic
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
| | - Jaromir Vasicek
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
- Research Centre AgroBioTech, Slovak University of Agriculture; Nitra Slovak Republic
| | - Barbora Kulikova
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
| | - Miroslav Bauer
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
- Faculty of Natural Sciences; Constantine the Philosopher University; Nitra Slovak republic
| | - Jozef Curlej
- Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture; Nitra Slovak Republic
| | - Andrej Balazi
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
| | - Peter Chrenek
- Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture; Nitra Slovak Republic
- Research Inst. for Animal Production, National Agricultural and Food Centre; Lužianky Slovak Republic
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25
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Abstract
The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining their pluripotent properties makes them a powerful tool for use in research, and provides tremendous potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent on the ability to effectively expand them in long-term culture while preserving their distinct nature. Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells to alternative platforms. These protocols have been used widely by laboratories around the world to successfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.
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Affiliation(s)
- Jennifer L Brehm
- WiCell Research Institute, 504 S. Rosa Rd., Suite 101, Madison, WI, 53719, USA
| | - Tenneille E Ludwig
- WiCell Research Institute, 504 S. Rosa Rd., Suite 101, Madison, WI, 53719, USA.
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26
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Shroff G, Vatsa D. Cell Viability of Human Embryonic Stem Cells Stored for a Period of 9 Years. EXP CLIN TRANSPLANT 2016; 15:344-349. [PMID: 27938317 DOI: 10.6002/ect.2016.0097] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES Human embryonic stem cells are pluripotent cell lines usually derived from human blastocysts. Their potential critically depends on long-term proliferative capacity, developmental potential after prolonged culture, and karyotypic stability. Cell viability is an important parameter for assessing cell sample quality. Here, we elaborate the stored human embryonic stem cell lines' viability in a ready to use form for a period of 9 years (from 2007 to 2015). MATERIALS AND METHODS Spare pre implantation stage in vitro fertilized ovum-derived cell lines were cultured in suitable media. Thereafter, they were centrifuged at 1000 revolutions/min over 5 minutes, and pellets were suspended in normal saline. Next, they were tested for viability from storage at -20°C. After being allowed to thaw slowly, the cells were stained with propidium iodide and analyzed using flow cytometry. Images of cells were taken at ×40 and ×100 magnification. RESULTS At ×100 magnification, cell population size ranged from 0.2 to 2 μm. The percentage of live cells was more than 95% throughout the 9 years. Cells frozen in 2015 showed cell viability of 96.8%. CONCLUSIONS We observed high cell viability in our cell lines for 9 years. Human embryonic stem cell lines in a ready-to-use form can be preserved for long-term purposes. Thus, they could be made available globally.
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27
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Mantle JL, Min L, Lee KH. Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model. Mol Pharm 2016; 13:4191-4198. [PMID: 27934481 DOI: 10.1021/acs.molpharmaceut.6b00818] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm2. By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.
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Affiliation(s)
- Jennifer L Mantle
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
| | - Lie Min
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
| | - Kelvin H Lee
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
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28
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Chen X, Niu W, Wang F, Yu W, Dai S, Kong H, Shu Y, Sun Y. Derivation of normal diploid human embryonic stem cells from tripronuclear zygotes with analysis of their copy number variation and loss of heterozygosity. Mol Reprod Dev 2016; 82:344-55. [PMID: 25988573 DOI: 10.1002/mrd.22485] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2014] [Accepted: 03/03/2015] [Indexed: 11/09/2022]
Abstract
This study sought to establish archives of genetic copy number variation (CNV) in human embryonic stem cell (hESC) lines that are associated with known diseases. We collected patients' fresh, discarded zygotes from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) protocols. A total of 208 fresh, tripronuclear, discarded zygotes were also collected in this study from patients on the third day of their treatment cycle, prior to transfer. The blastula-formation rates were 13.51% (26/192) and 26.7% (4/15) while the high-quality blastocyst formation rates were 5.8% (11/192) and 20% (3/15) in the IVF and ICSI groups, respectively. The inner cell mass (ICM) from each embryo was mechanically separated, and then grown on feeder layers consisting of mouse embryonic fibroblasts and human foreskin fibroblasts (a 1:1 mixture). The hESC karyotype was determined by traditional G-banding; analysis of the results for the Zh19P25 and Zh20P24 cell lines showed that both were 46 XY. CNV and loss-of-heterozygosity analysis of hESC gDNA was performed to assess the genetic characteristics associated with molecular diseases using the high-resolution Infinium High-Density HumanCytoSNP-12 DNA chip. Seven CNVs in Zh19P25 and Zh20P24 were deletions, and a region that corresponds to Potocki-Shaffer disease, 11p11.2-11p11.12 in Zh20P24, showed a 2.98-Mb loss. These data together suggest that single-nucleotide polymorphism (SNP) microarray analysis for molecular cytogenetic features can help to distinguish hESC lines with a normal karyotype from tripronuclear zygotes with known, disease-related characteristics.
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Affiliation(s)
- Xuemei Chen
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.,Department of Human Anatomy, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
| | - Wenbin Niu
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Fang Wang
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Wenzhu Yu
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Shanjun Dai
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Huijuan Kong
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Yimin Shu
- Department of Obstetrics and Gynecology, Stanford University Medical Center, Palo Alto, California
| | - Yingpu Sun
- Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
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29
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Di Foggia V, Makwana P, Ali RR, Sowden JC. Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement. J Ocul Pharmacol Ther 2016; 32:240-52. [PMID: 27027805 DOI: 10.1089/jop.2015.0143] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Stem cell therapies are being explored as potential treatments for retinal disease. How to replace neurons in a degenerated retina presents a continued challenge for the regenerative medicine field that, if achieved, could restore sight. The major issues are: (i) the source and availability of donor cells for transplantation; (ii) the differentiation of stem cells into the required retinal cells; and (iii) the delivery, integration, functionality, and survival of new cells in the host neural network. This review considers the use of induced pluripotent stem cells (iPSC), currently under intense investigation, as a platform for cell transplantation therapy. Moreover, patient-specific iPSC are being developed for autologous cell transplantation and as a tool for modeling specific retinal diseases, testing gene therapies, and drug screening.
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Affiliation(s)
- Valentina Di Foggia
- 1 UCL Institute of Child Health, University College London , London, United Kingdom
| | - Priyanka Makwana
- 1 UCL Institute of Child Health, University College London , London, United Kingdom
| | - Robin R Ali
- 2 UCL Institute of Ophthalmology , London, United Kingdom
| | - Jane C Sowden
- 1 UCL Institute of Child Health, University College London , London, United Kingdom
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30
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Asprer JST, Lakshmipathy U. Current methods and challenges in the comprehensive characterization of human pluripotent stem cells. Stem Cell Rev Rep 2016; 11:357-72. [PMID: 25504379 DOI: 10.1007/s12015-014-9580-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Pluripotent stem cells (PSCs) are powerful tools for basic scientific research and promising agents for drug discovery and regenerative medicine. Technological advances have made it increasingly easy to generate PSCs but the various lines generated may differ in their characteristics based on their origin, derivation, number of passages, and culture conditions. In order to confirm the pluripotency, quality, identity, and safety of pluripotent cell lines as they are derived and maintained, it is critical to perform a panel of characterization assays. Functional pluripotency is determined using tests that rely on the expression of specific markers in the undifferentiated and differentiated states; tests for quality, identity and safety are less specialized. This article provides a comprehensive review of current practices in PSC characterization and explores challenges in the field, from the selection of markers to the development of simple and scalable methods. It also delves into emerging trends like the adoption of alternative assays that could be used to supplement or replace traditional methods, specifically the use of in silico assays for determining pluripotency.
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Affiliation(s)
- Joanna S T Asprer
- Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA, 92008, USA
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31
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Rebuzzini P, Zuccotti M, Redi CA, Garagna S. Chromosomal Abnormalities in Embryonic and Somatic Stem Cells. Cytogenet Genome Res 2015; 147:1-9. [PMID: 26583376 DOI: 10.1159/000441645] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/30/2015] [Indexed: 12/20/2022] Open
Abstract
The potential use of stem cells (SCs) for tissue engineering, regenerative medicine, disease modeling, toxicological studies, drug delivery, and as in vitro model for the study of basic developmental processes implies large-scale in vitro culture. Here, after a brief description of the main techniques used for karyotype analysis, we will give a detailed overview of the chromosome abnormalities described in pluripotent (embryonic and induced pluripotent SCs) and somatic SCs, and the possible causes of their origin during culture.
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Affiliation(s)
- Paola Rebuzzini
- Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie, Universitx00E0; degli Studi di Pavia, Pavia, Italy
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32
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Badur MG, Zhang H, Metallo CM. Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance. Biotechnol J 2015; 10:1600-11. [PMID: 26289220 DOI: 10.1002/biot.201400749] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Revised: 07/10/2015] [Accepted: 08/18/2015] [Indexed: 12/26/2022]
Abstract
To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications.
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Affiliation(s)
- Mehmet G Badur
- Department of Bioengineering, University of California, San Diego, La Jolla, USA
| | - Hui Zhang
- Department of Bioengineering, University of California, San Diego, La Jolla, USA
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33
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Kim YE, Park JA, Ha YW, Park SK, Kim HS, Oh SK, Lee Y. Chromosomal Modification in Human Embryonic Stem Cells Cultured in a Feeder-Free Condition after Single Cell Dissociation using Accutase. Dev Reprod 2015; 16:353-61. [PMID: 25949110 PMCID: PMC4282239 DOI: 10.12717/dr.2012.16.4.353] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2012] [Revised: 11/19/2012] [Accepted: 12/13/2012] [Indexed: 12/24/2022]
Abstract
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.
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Affiliation(s)
- Young-Eun Kim
- Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea ; Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea
| | - Jeong-A Park
- Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea
| | - Yang-Wha Ha
- Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea
| | - Sang-Kyu Park
- Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea
| | - Hee Sun Kim
- Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul 110-799, Korea ; IVF Laboratory, Dept. of Obstetrics and Gynecology, Seoul National University Hospital, Seoul 110-744, Korea
| | - Sun Kyung Oh
- Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul 110-799, Korea ; IVF Laboratory, Dept. of Obstetrics and Gynecology, Seoul National University Hospital, Seoul 110-744, Korea
| | - Younghee Lee
- Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea ; Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea
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34
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Tosca L, Feraud O, Magniez A, Bas C, Griscelli F, Bennaceur-Griscelli A, Tachdjian G. Genomic instability of human embryonic stem cell lines using different passaging culture methods. Mol Cytogenet 2015; 8:30. [PMID: 26052346 PMCID: PMC4456787 DOI: 10.1186/s13039-015-0133-8] [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: 01/07/2015] [Accepted: 04/07/2015] [Indexed: 12/22/2022] Open
Abstract
Background Human embryonic stem cells exhibit genomic instability that can be related to culture duration or to the passaging methods used for cell dissociation. In order to study the impact of cell dissociation techniques on human embryonic stem cells genomic instability, we cultured H1 and H9 human embryonic stem cells lines using mechanical/manual or enzymatic/collagenase-IV dissociation methods. Genomic instability was evaluated at early (<p60) and late (>p60) passages by using oligonucleotide based array-comparative genomic hybridization 105 K with a mean resolution of 50 Kb. Results DNA variations were mainly located on subtelomeric and pericentromeric regions with sizes <100 Kb. In this study, 9 recurrent genomic variations were acquired during culture including the well known duplication 20q11.21. When comparing cell dissociation methods, we found no significant differences between DNA variations number and size, DNA gain or DNA loss frequencies, homozygous loss frequencies and no significant difference on the content of genes involved in development, cell cycle tumorigenesis and syndrome disease. In addition, we have never found any malignant tissue in 4 different teratoma representative of the two independent stem cell lines. Conclusions These results show that the occurrence of genomic instability in human embryonic stem cells is similar using mechanical or collagenase IV-based enzymatic cell culture dissociation methods. All the observed genomic variations have no impact on the development of malignancy. Electronic supplementary material The online version of this article (doi:10.1186/s13039-015-0133-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Lucie Tosca
- AP-HP, Histologie-Embryologie-Cytogénétique, Hôpitaux Universitaires Paris Sud, Clamart, F-92141 France ; Université Paris Sud, Le Kremlin-Bicêtre, F-94275 France ; Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France
| | - Olivier Feraud
- Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France
| | - Aurélie Magniez
- Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France
| | - Cécile Bas
- AP-HP, Histologie-Embryologie-Cytogénétique, Hôpitaux Universitaires Paris Sud, Clamart, F-92141 France ; Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France
| | - Frank Griscelli
- Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France ; Université Paris Descartes, Sorbonne Paris Cité, F-75006 France
| | - Annelise Bennaceur-Griscelli
- Université Paris Sud, Le Kremlin-Bicêtre, F-94275 France ; Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France ; AP-HP, Hématologie, Hôpitaux Universitaires Paris Sud, Villejuif, F-94801 France
| | - Gérard Tachdjian
- AP-HP, Histologie-Embryologie-Cytogénétique, Hôpitaux Universitaires Paris Sud, Clamart, F-92141 France ; Université Paris Sud, Le Kremlin-Bicêtre, F-94275 France ; Esteam Paris Sud INSERM UMR-S 935, Villejuif, F-94801 France
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35
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Concise Review: Methods and Cell Types Used to Generate Down Syndrome Induced Pluripotent Stem Cells. J Clin Med 2015; 4:696-714. [PMID: 26239351 PMCID: PMC4470162 DOI: 10.3390/jcm4040696] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2015] [Revised: 03/23/2015] [Accepted: 03/31/2015] [Indexed: 01/29/2023] Open
Abstract
Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Several models have been used to investigate the mechanisms by which the extra copy of chromosome 21 leads to the DS phenotype. In the last five years, several laboratories have been successful in reprogramming patient cells carrying the trisomy 21 anomaly into induced pluripotent stem cells, i.e., T21-iPSCs. In this review, we summarize the different T21-iPSCs that have been generated with a particular interest in the technical procedures and the somatic cell types used for the reprogramming.
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Fan HC, Ho LI, Chi CS, Cheng SN, Juan CJ, Chiang KL, Lin SZ, Harn HJ. Current proceedings of cerebral palsy. Cell Transplant 2015; 24:471-85. [PMID: 25706819 DOI: 10.3727/096368915x686931] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Cerebral palsy (CP) is a complicated disease with varying causes and outcomes. It has created significant burden to both affected families and societies, not to mention the quality of life of the patients themselves. There is no cure for the disease; therefore, development of effective therapeutic strategies is in great demand. Recent advances in regenerative medicine suggest that the transplantation of stem cells, including embryonic stem cells, neural stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, umbilical cord blood cells, and human embryonic germ cells, focusing on the root of the problem, may provide the possibility of developing a complete cure in treating CP. However, safety is the first factor to be considered because some stem cells may cause tumorigenesis. Additionally, more preclinical and clinical studies are needed to determine the type of cells, route of delivery, cell dose, timing of transplantation, and combinatorial strategies to achieve an optimal outcome.
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Affiliation(s)
- Hueng-Chuen Fan
- Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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Simonson OE, Domogatskaya A, Volchkov P, Rodin S. The safety of human pluripotent stem cells in clinical treatment. Ann Med 2015; 47:370-80. [PMID: 26140342 DOI: 10.3109/07853890.2015.1051579] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) have practically unlimited proliferation potential and a capability to differentiate into any cell type in the human body. Since the first derivation in 1998, they have been an attractive source of cells for regenerative medicine. Numerous ethical, technological, and regulatory complications have been hampering hPSC use in clinical applications. Human embryonic stem cells (ESCs), parthenogenetic human ESCs, human nuclear transfer ESCs, and induced pluripotent stem cells are four types of hPSCs that are different in many clinically relevant features such as propensity to epigenetic abnormalities, generation methods, and ability for development of autologous cell lines. Propensity to genetic mutations and tumorigenicity are common features of all pluripotent cells that complicate hPSC-based therapies. Several recent advances in methods of derivation, culturing, and monitoring of hPSCs have addressed many ethical concerns and technological challenges in development of clinical-grade hPSC lines. Generation of banks of such lines may be useful to minimize immune rejection of hPSC-derived allografts. In this review, we discuss different sources of hPSCs available at the moment, various safety risks associated with them, and possible solutions for successful use of hPSCs in the clinic. We also discuss ongoing clinical trials of hPSC-based treatments.
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Affiliation(s)
- Oscar E Simonson
- a Division of Cardiothoracic Surgery and Anesthesiology, Department of Molecular Medicine and Surgery , Karolinska Institutet, Karolinska University Hospital , 171 77 Stockholm , Sweden
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Zhao P, Schulz TC, Sherrer ES, Weatherly DB, Robins AJ, Wells L. The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry. Proteomics 2014; 15:554-66. [PMID: 25367160 DOI: 10.1002/pmic.201400132] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2014] [Revised: 09/22/2014] [Accepted: 10/28/2014] [Indexed: 01/06/2023]
Abstract
Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked β-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future.
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Affiliation(s)
- Peng Zhao
- Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
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Human mesenchymal stem cells possess different biological characteristics but do not change their therapeutic potential when cultured in serum free medium. Stem Cell Res Ther 2014; 5:132. [PMID: 25476802 PMCID: PMC4445567 DOI: 10.1186/scrt522] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Introduction Mesenchymal stem cells (MSCs) are widely investigated in clinical researches to treat various diseases. Classic culture medium for MSCs, even for clinical use, contains fetal bovine serum. The serum-containing medium (SCM) seems a major obstacle for MSCs-related therapies due to the risk of contamination of infectious pathogens. Some studies showed that MSCs could be expanded in serum free medium (SFM); however, whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered. Methods Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in a chemical defined serum free medium. Growth, multipotency, surface antigen expression, telomerase, immunosuppressive ability, gene expression profile and genomic stability of hUC-MSCs cultured in SFM and SCM were analyzed and compared side by side. Results hUC-MSCs propagated more slowly and senesce ultimately in SFM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in growth rate, telomerase, gene expression profile. However, SFM-expanded hUC-MSCs maintained multipotency and the profile of surface antigen which were used to define human MSCs. Both SFM- and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture. Conclusion hUC-MCSs could be expanded in SFM safely to obtain enough cells for clinical application, meeting the basic criteria for human mesenchymal stem cells. hUC-MSCs cultured in SFM were distinct from hUC-MSCs cultured in SCM, yet they remained therapeutic potentials for future regenerative medicine. Electronic supplementary material The online version of this article (doi:10.1186/scrt522) contains supplementary material, which is available to authorized users.
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Bai Q, Ramirez JM, Becker F, Pantesco V, Lavabre-Bertrand T, Hovatta O, Lemaître JM, Pellestor F, De Vos J. Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells. Stem Cells Dev 2014; 24:653-62. [PMID: 25254421 DOI: 10.1089/scd.2014.0292] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Simplified culture conditions are essential for large-scale drug screening and medical applications of human pluripotent stem cells (hPSCs). However, hPSCs [ie, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (iPSCs) are prone to genomic instability, a phenomenon that is highly influenced by the culture conditions. Enzymatic dissociation, a cornerstone of large-scale hPSC culture systems, has been reported to be deleterious, but the extent and the timeline of the genomic alterations induced by this passaging technique are still unclear. We prospectively monitored three hESC lines that were initially derived and cultured on human feeders and passaged mechanically before switching to enzymatic single-cell passaging. We show that karyotype abnormalities and copy number variations are not restricted to long-term culture, but can occur very rapidly, within five passages after switching hESCs to enzymatic dissociation. Subchromosomal abnormalities preceded or accompanied karyotype abnormalities and were associated with increased occurrence of DNA double-strand breaks. Our results indicate that enzymatic single-cell passaging can be highly deleterious to the hPSC genome, even when used only for a limited period of time. Moreover, hPSC culture techniques should be reappraised by complementing the routine karyotype analysis with more sensitive techniques, such as microarrays, to detect subchromosomal abnormalities.
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Affiliation(s)
- Qiang Bai
- 1 INSERM , U1040, Montpellier, France
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Liu Y, Wu D, Lao D, Peterson C, Hohenstein Elliott KA, Snyder EY. Laser‐Assisted Generation of Human Induced Pluripotent Stem Cells. ACTA ACUST UNITED AC 2014; 31:4A.7.1-15. [DOI: 10.1002/9780470151808.sc04a07s31] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Affiliation(s)
- Yang Liu
- Stem Cell Research Center and Core Facility, Sanford‐Burnham Medical Research Institute La Jolla California
| | - Dongmei Wu
- Stem Cell Research Center and Core Facility, Sanford‐Burnham Medical Research Institute La Jolla California
| | - Donglin Lao
- Stem Cell Research Center and Core Facility, Sanford‐Burnham Medical Research Institute La Jolla California
| | - Cory Peterson
- Cell Engineering Unit, Intrexon Corporation San Diego California
| | | | - Evan Y. Snyder
- Sanford‐Burnham Medical Research Institute La Jolla California
- Sanford Consortium for Regenerative Medicine La Jolla California
- Department of Pediatrics, University of California at San Diego La Jolla California
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Oliveira PH, da Silva CL, Cabral JM. Concise Review: Genomic Instability in Human Stem Cells: Current Status and Future Challenges. Stem Cells 2014; 32:2824-32. [DOI: 10.1002/stem.1796] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2014] [Revised: 06/03/2014] [Accepted: 06/09/2014] [Indexed: 12/26/2022]
Affiliation(s)
- Pedro H. Oliveira
- Institut Pasteur; Microbial Evolutionary Genomics, Département Génomes et Génétique; Paris France
- CNRS; UMR3525 Paris France
| | - Cláudia Lobato da Silva
- Institute for Biotechnology and Bioengineering, Department of Bioengineering; Instituto Superior Técnico, Universidade de Lisboa; Lisboa Portugal
| | - Joaquim M.S. Cabral
- Institute for Biotechnology and Bioengineering, Department of Bioengineering; Instituto Superior Técnico, Universidade de Lisboa; Lisboa Portugal
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Xie P, Sun Y, Ouyang Q, Hu L, Tan Y, Zhou X, Xiong B, Zhang Q, Yuan D, Pan Y, Liu T, Liang P, Lu G, Lin G. Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture. Stem Cells 2014; 32:391-401. [PMID: 24123616 DOI: 10.1002/stem.1558] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2013] [Revised: 07/28/2013] [Accepted: 08/25/2013] [Indexed: 12/18/2022]
Abstract
Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs.
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Affiliation(s)
- Pingyuan Xie
- Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha, China; Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha, China
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Shelley BC, Gowing G, Svendsen CN. A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue. J Vis Exp 2014. [PMID: 24962813 DOI: 10.3791/51219] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
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Aneuploidy in pluripotent stem cells and implications for cancerous transformation. Protein Cell 2014; 5:569-79. [PMID: 24899134 PMCID: PMC4130921 DOI: 10.1007/s13238-014-0073-9] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Accepted: 04/30/2014] [Indexed: 12/11/2022] Open
Abstract
Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.
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Zhou D, Lin G, Zeng SC, Xiong B, Xie PY, Cheng DH, Zheng Q, Ouyang Q, Zhou XY, Tang WL, Sun Y, Lu GY, Lu GX. Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells. Arch Toxicol 2014; 89:33-45. [PMID: 24838295 DOI: 10.1007/s00204-014-1250-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2013] [Accepted: 04/15/2014] [Indexed: 02/07/2023]
Abstract
How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.
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Affiliation(s)
- Di Zhou
- Institute of Reproduction and Stem Cell Engineering, Central South University, 8 Luyun Road, Changsha, 410000, Hunan, China
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Filioli Uranio M, Dell'Aquila ME, Caira M, Guaricci AC, Ventura M, Catacchio CR, Martino NA, Valentini L. Characterization and in vitro differentiation potency of early-passage canine amnion- and umbilical cord-derived mesenchymal stem cells as related to gestational age. Mol Reprod Dev 2014; 81:539-51. [PMID: 24659564 DOI: 10.1002/mrd.22322] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2013] [Accepted: 03/15/2014] [Indexed: 12/20/2022]
Abstract
Fetal adnexa are a non-controversial source of mesenchymal stem cells (MSCs) that have high plasticity, a high proliferation rate, and the ability to differentiate towards multiple lineages. MSC populations have been characterized for their stemness and differentiation capabilities; more recent work has focused on MSC selection and on establishing predictable elements to discriminate the cells with the most potential for regenerative medicine. In this study, we cytogenetically and molecularly characterized and followed the in vitro proliferation and differentiation potential of early-passage canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) isolated from fetuses at early (35-40 days) and late (45-55 days) gestational ages. We found that cells from both fetal gestational ages showed similar features. In all examined cell lines, the morphology of proliferating cells typically appeared fibroblast-like. Population doublings, passaged up to 10 times, increased significantly with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age at early passages. Passage-3 AM- and UCM-MSCs from both gestational phases also expressed embryonic (POU5F1) and mesenchymal (CD29, CD44) stemness markers, whereas hematopoietic and histocompatibility markers were never found in any sample. Passage-3 cell populations of each cell type were also multipotential as they could differentiate into neurocytes and osteocytes, based on cell morphology, specific stains, and molecular analysis. These results indicated that MSCs retrieved from the UCM and AM in the early and late fetal phases of gestation could be used for canine regenerative medicine.
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Affiliation(s)
- Manuel Filioli Uranio
- Veterinary Clinics and Animal Productions Section, Department for Emergency and Organ Transplantation, University of Bari Aldo Moro, Valenzano, Italy
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Borgonovo T, Vaz IM, Senegaglia AC, Rebelatto CLK, Brofman PRS. Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center. Rev Bras Hematol Hemoter 2014; 36:202-7. [PMID: 25031060 PMCID: PMC4109748 DOI: 10.1016/j.bjhh.2014.03.006] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2013] [Accepted: 01/14/2014] [Indexed: 12/31/2022] Open
Abstract
Objective To present the initial results of first three years of implementation of a genetic evaluation test for bone marrow-derived mesenchymal stem cells in a Cell Technology Center. Methods A retrospective study was carried out of 21 candidates for cell therapy. After the isolation of bone marrow mononuclear cells by density gradient, mesenchymal stem cells were cultivated and expanded at least until the second passage. Cytogenetic analyses were performed before and after cell expansion (62 samples) using G-banded karyotyping. Results All the samples analyzed, before and after cell expansion, had normal karyotypes, showing no clonal chromosomal changes. Signs of chromosomal instability were observed in 11 out of 21 patients (52%). From a total of 910 analyzed metaphases, five chromatid gaps, six chromatid breaks and 14 tetraploid cells were detected giving as total of 25 metaphases with chromosome damage (2.75%). Conclusion The absence of clonal chromosomal aberrations in our results for G-banded karyotyping shows the maintenance of chromosomal stability of bone marrow-derived mesenchymal stem cells until the second passage; however, signs of chromosomal instability such as chromatid gaps, chromosome breaks and tetraploidy indicate that the long-term cultivation of these cells can provide an intermediate step for tumorigenesis.
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Affiliation(s)
- Tamara Borgonovo
- Pontifícia Universidade Católica do Paraná (PUC-PR), Curitiba, PR, Brazil.
| | - Isadora May Vaz
- Pontifícia Universidade Católica do Paraná (PUC-PR), Curitiba, PR, Brazil
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Rajamani K, Li YS, Hsieh DK, Lin SZ, Harn HJ, Chiou TW. Genetic and epigenetic instability of stem cells. Cell Transplant 2014; 23:417-33. [PMID: 24622296 DOI: 10.3727/096368914x678472] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Recently, research on stem cells has been receiving an increasing amount of attention, both for its advantages and disadvantages. Genetic and epigenetic instabilities among stem cells have been a recurring obstacle to progress in regenerative medicine using stem cells. Various reports have stated that these instabilities can transform stem cells when transferred in vivo and thus have the potential to develop tumors. Previous research has shown that various extrinsic and intrinsic factors can contribute to the stability of stem cells. The extrinsic factors include growth supplements, growth factors, oxygen tension, passage technique, and cryopreservation. Controlling these factors based on previous reports may assist researchers in developing strategies for the production and clinical application of "safe" stem cells. On the other hand, the intrinsic factors can be unpredictable and uncontrollable; therefore, to ensure the successful use of stem cells in regenerative medicine, it is imperative to develop and implement appropriate strategies and technique for culturing stem cells and to confirm the genetic and epigenetic safety of these stem cells before employing them in clinical trials.
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Affiliation(s)
- Karthyayani Rajamani
- Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan
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Sundberg M, Isacson O. Advances in stem-cell–generated transplantation therapy for Parkinson's disease. Expert Opin Biol Ther 2014; 14:437-53. [DOI: 10.1517/14712598.2014.876986] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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