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1
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Lewis PA, Silajdžić E, Smith H, Bates N, Smith CA, Mancini FE, Knight D, Denning C, Brison DR, Kimber SJ. A secreted proteomic footprint for stem cell pluripotency. PLoS One 2024; 19:e0299365. [PMID: 38875182 PMCID: PMC11178176 DOI: 10.1371/journal.pone.0299365] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Accepted: 02/08/2024] [Indexed: 06/16/2024] Open
Abstract
With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.
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Affiliation(s)
- Philip A. Lewis
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Edina Silajdžić
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Helen Smith
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Nicola Bates
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Christopher A. Smith
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Fabrizio E. Mancini
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - David Knight
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Chris Denning
- Biodiscovery Institute, Division of Cancer & Stem Cells, School of Medicine, University of Nottingham, University Park, Nottingham, United Kingdom
| | - Daniel R. Brison
- Royal Manchester Children’s Hospital, Manchester, United Kingdom
| | - Susan J. Kimber
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
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2
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Kidder BL. Decoding the universal human chromatin landscape through teratoma-based profiling. Nucleic Acids Res 2024; 52:3589-3606. [PMID: 38281248 PMCID: PMC11039989 DOI: 10.1093/nar/gkae021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 12/15/2023] [Accepted: 01/04/2024] [Indexed: 01/30/2024] Open
Abstract
Teratoma formation is key for evaluating differentiation of human pluripotent stem cells into embryonic germ layers and serves as a model for understanding stem cell differentiation and developmental processes. Its potential for insights into epigenome and transcriptome profiling is significant. This study integrates the analysis of the epigenome and transcriptome of hESC-generated teratomas, comparing transcriptomes between hESCs and teratomas. It employs cell type-specific expression patterns from single-cell data to deconvolve RNA-Seq data and identify cell types within teratomas. Our results provide a catalog of activating and repressive histone modifications, while also elucidating distinctive features of chromatin states. Construction of an epigenetic signature matrix enabled the quantification of diverse cell populations in teratomas and enhanced the ability to unravel the epigenetic landscape in heterogeneous tissue contexts. This study also includes a single cell multiome atlas of expression (scRNA-Seq) and chromatin accessibility (scATAC-Seq) of human teratomas, further revealing the complexity of these tissues. A histology-based digital staining tool further complemented the annotation of cell types in teratomas, enhancing our understanding of their cellular composition. This research is a valuable resource for examining teratoma epigenomic and transcriptomic landscapes and serves as a model for epigenetic data comparison.
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Affiliation(s)
- Benjamin L Kidder
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA
- Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA
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3
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Lo EKW, Velazquez JJ, Peng D, Kwon C, Ebrahimkhani MR, Cahan P. Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols. Stem Cell Reports 2023; 18:1721-1742. [PMID: 37478860 PMCID: PMC10444577 DOI: 10.1016/j.stemcr.2023.06.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Revised: 06/16/2023] [Accepted: 06/17/2023] [Indexed: 07/23/2023] Open
Abstract
Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.
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Affiliation(s)
- Emily K W Lo
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Jeremy J Velazquez
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Da Peng
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Chulan Kwon
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Mo R Ebrahimkhani
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
| | - Patrick Cahan
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
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4
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Suresh Babu S, Duvvuru H, Baker J, Switalski S, Shafa M, Panchalingam KM, Dadgar S, Beller J, Ahmadian Baghbaderani B. Characterization of human induced pluripotent stems cells: Current approaches, challenges, and future solutions. BIOTECHNOLOGY REPORTS (AMSTERDAM, NETHERLANDS) 2023; 37:e00784. [PMID: 36818379 PMCID: PMC9929203 DOI: 10.1016/j.btre.2023.e00784] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 01/09/2023] [Accepted: 01/19/2023] [Indexed: 01/25/2023]
Abstract
Human induced pluripotent stem cells (iPSC) have demonstrated massive potentials for use in regenerative and personalized medicine due to their ability to expand in culture and differentiate into specialized cells with therapeutic benefits. However, in order to industrialize iPSC-derived therapies, it is necessary to address the existing challenges surrounding the analytics implemented in the manufacturing process to evaluate and monitor cell expansion, differentiation, and quality of the final products. Here, we review some of the key analytical methods used as part of identity, potency, or safety for in-process or final product release testing and highlighted the challenges and potential solutions for consideration in the Chemistry, Manufacturing and Controls (CMC) strategy for iPSC-based therapies. Some of the challenges associated with characterization and testing of iPSC-based products are related to the choice of analytical technology (to ensure fit-for-purpose), assay reliability and robustness. Automation of analytical methods may be required to reduce hands on time, and improve reliability of the methods through reducing assay variability. Indeed, we have shown that automation of analytical methods is feasible (evaluated using an ELISA based assay) and would result in more precise measurements (demonstrated by lower co-efficient of Variation and standard deviation), less hands-on time, and swift compared to a manually run assay. Therefore, in order to support commercialization of iPSC-based therapies we suggest a well-designed testing strategy to be established in the development phase while incorporating robust, reproducible, reliable, and potentially automated analytics in the manufacturing process.
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5
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Andrews PW, Barbaric I, Benvenisty N, Draper JS, Ludwig T, Merkle FT, Sato Y, Spits C, Stacey GN, Wang H, Pera MF. The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells. Cell Stem Cell 2022; 29:1624-1636. [PMID: 36459966 DOI: 10.1016/j.stem.2022.11.006] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 11/08/2022] [Accepted: 11/08/2022] [Indexed: 12/05/2022]
Abstract
It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.
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Affiliation(s)
- Peter W Andrews
- Centre for Stem Cell Biology, School of Biological Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK; Steering Committee, International Stem Cell Initiative
| | - Ivana Barbaric
- Centre for Stem Cell Biology, School of Biological Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK; Steering Committee, International Stem Cell Initiative
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel; Steering Committee, International Stem Cell Initiative
| | - Jonathan S Draper
- Stem Cell Network, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada; Steering Committee, International Stem Cell Initiative
| | - Tenneille Ludwig
- WiCell Research Institute, Madison, WI, USA; University of Wisconsin-Madison, Madison, WI 53719, USA; Steering Committee, International Stem Cell Initiative
| | - Florian T Merkle
- Wellcome Trust-Medical Research Council Institute of Metabolic Science, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0QQ, UK; Steering Committee, International Stem Cell Initiative
| | - Yoji Sato
- Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa 210-9501, Japan; Steering Committee, International Stem Cell Initiative
| | - Claudia Spits
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium; Steering Committee, International Stem Cell Initiative
| | - Glyn N Stacey
- International Stem Cell Banking Initiative, 2 High Street, Barley, UK; National Stem Cell Resource Centre, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100190, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China; Steering Committee, International Stem Cell Initiative
| | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101, Beijing, China; Beijing Institute for Stem Cell and Regenerative Medicine, 100101, Beijing, China; Steering Committee, International Stem Cell Initiative
| | - Martin F Pera
- The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA; Steering Committee, International Stem Cell Initiative.
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6
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Lezmi E, Benvenisty N. The Tumorigenic Potential of Human Pluripotent Stem Cells. Stem Cells Transl Med 2022; 11:791-796. [PMID: 35679163 PMCID: PMC9397652 DOI: 10.1093/stcltm/szac039] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 04/24/2022] [Indexed: 11/23/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are currently evaluated for clinical applications due to their proliferation and differentiation capacities, raising the need to both assess and enhance, the safety of hPSC-based treatments. Distinct molecular features contribute to the tumorigenicity of hPSCs, manifested in the formation of teratoma tumors upon transplantation in vivo. Prolonged in vitro culturing of hPSCs can enhance selection for specific genetic aberrations, either at the chromosome or gene level. Some of these aberrations are tightly linked to human tumor pathology and increase the tumorigenic aggressiveness of the abnormal cells. In this perspective, we describe major tumor-associated risk factors entailed in hPSC-based therapy, and present precautionary and safety measures relevant for the development and application of such therapies.
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Affiliation(s)
- Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel
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7
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Keller A, Krivec N, Markouli C, Spits C. Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells. Methods Mol Biol 2022; 2429:57-72. [PMID: 35507155 DOI: 10.1007/978-1-0716-1979-7_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Human pluripotent stem cells have a wide variety of potential applications, ranging from clinical translation to in vitro disease modeling. However, there is significant variation in the potential of individual cell lines to differentiate towards each of the three germ layers as a result of (epi)genetic background, culture conditions, and other factors. We describe here in detail a methodology to evaluate this bias using short directed differentiation towards neuroectoderm, mesendoderm, and definitive endoderm in combination with quantification by RT-qPCR and immunofluorescent stains.
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Affiliation(s)
- Alexander Keller
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Nuša Krivec
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Christina Markouli
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Claudia Spits
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium.
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8
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Andrews PW. Human pluripotent stem cells: tools for regenerative medicine. BIOMATERIALS TRANSLATIONAL 2021; 2:294-300. [PMID: 35837419 PMCID: PMC9255800 DOI: 10.12336/biomatertransl.2021.04.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Subscribe] [Scholar Register] [Received: 11/13/2021] [Revised: 12/12/2021] [Accepted: 12/20/2021] [Indexed: 01/17/2023]
Abstract
Human embryonic stem cells and induced pluripotent stem cells, together denoted as pluripotent stem cells have opened up unprecedented opportunities for developments in human healthcare over the past 20 years. Although much about the properties and behaviour of these cells required to underpin their applications has been discovered over this time, a number of issues remain. This brief review considers the history of these developments and some of the underlying biology, pointing out some of the problems still to be resolved, particularly in relation to their genetic stability and possible malignancy.
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Affiliation(s)
- Peter W. Andrews
- The Centre for Stem Cell Biology, The School of Bioscience, The University of Sheffield, Sheffield, UK
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9
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Vickers A, Tewary M, Laddach A, Poletti M, Salameti V, Fraternali F, Danovi D, Watt FM. Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation. Stem Cell Reports 2021; 16:2628-2641. [PMID: 34678211 PMCID: PMC8581167 DOI: 10.1016/j.stemcr.2021.09.017] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Revised: 09/22/2021] [Accepted: 09/23/2021] [Indexed: 12/03/2022] Open
Abstract
Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation.
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Affiliation(s)
- Alice Vickers
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Mukul Tewary
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Anna Laddach
- Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, Great Maze Pond, London SE1 9RT, UK; Development and Homeostasis of the Nervous System Laboratory, The Francis Crick Institute, London NW1 1AT, UK
| | - Martina Poletti
- Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK; Quadram Institute, Norwich Research Park, Norwich NR4 7UZ, UK
| | - Vasiliki Salameti
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Franca Fraternali
- Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, Great Maze Pond, London SE1 9RT, UK
| | - Davide Danovi
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK; bit.bio, Babraham Research Campus, The Dorothy Hodgkin Building, Cambridge CB22 3FH, UK
| | - Fiona M Watt
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK.
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10
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Gouin KH, Ing N, Plummer JT, Rosser CJ, Ben Cheikh B, Oh C, Chen SS, Chan KS, Furuya H, Tourtellotte WG, Knott SRV, Theodorescu D. An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer. Nat Commun 2021; 12:4906. [PMID: 34385456 PMCID: PMC8361097 DOI: 10.1038/s41467-021-25103-7] [Citation(s) in RCA: 93] [Impact Index Per Article: 23.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 07/22/2021] [Indexed: 12/20/2022] Open
Abstract
Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.
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Affiliation(s)
- Kenneth H Gouin
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Nathan Ing
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Jasmine T Plummer
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Charles J Rosser
- Department of Surgery (Urology), Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA
| | - Bassem Ben Cheikh
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Catherine Oh
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Stephanie S Chen
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Keith Syson Chan
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA
- Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Hideki Furuya
- Department of Surgery (Urology), Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA
| | - Warren G Tourtellotte
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA
- Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA
- Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Simon R V Knott
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
- Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA.
| | - Dan Theodorescu
- Department of Surgery (Urology), Cedars-Sinai Medical Center, Los Angeles, CA, USA.
- Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA.
- Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
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11
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Dziedzicka D, Tewary M, Keller A, Tilleman L, Prochazka L, Östblom J, Couvreu De Deckersberg E, Markouli C, Franck S, Van Nieuwerburgh F, Spits C, Zandstra PW, Sermon K, Geens M. Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm. Sci Rep 2021; 11:6137. [PMID: 33731744 PMCID: PMC7969605 DOI: 10.1038/s41598-021-85447-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 02/23/2021] [Indexed: 02/06/2023] Open
Abstract
Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.
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Affiliation(s)
- Dominika Dziedzicka
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mukul Tewary
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada ,grid.13097.3c0000 0001 2322 6764Centre for Stem Cells and Regenerative Medicine, King’s College London, Guy’s Hospital, London, SE1 9RT UK
| | - Alexander Keller
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Laurentijn Tilleman
- grid.5342.00000 0001 2069 7798Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Ghent, Belgium
| | - Laura Prochazka
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada
| | - Joel Östblom
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada
| | - Edouard Couvreu De Deckersberg
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Christina Markouli
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Silvie Franck
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Filip Van Nieuwerburgh
- grid.5342.00000 0001 2069 7798Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Ghent, Belgium
| | - Claudia Spits
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Peter W. Zandstra
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada ,grid.17091.3e0000 0001 2288 9830Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4 Canada ,grid.17091.3e0000 0001 2288 9830School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3 Canada
| | - Karen Sermon
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mieke Geens
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
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12
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Cahan P, Cacchiarelli D, Dunn SJ, Hemberg M, de Sousa Lopes SMC, Morris SA, Rackham OJL, Del Sol A, Wells CA. Computational Stem Cell Biology: Open Questions and Guiding Principles. Cell Stem Cell 2021; 28:20-32. [PMID: 33417869 PMCID: PMC7799393 DOI: 10.1016/j.stem.2020.12.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Computational biology is enabling an explosive growth in our understanding of stem cells and our ability to use them for disease modeling, regenerative medicine, and drug discovery. We discuss four topics that exemplify applications of computation to stem cell biology: cell typing, lineage tracing, trajectory inference, and regulatory networks. We use these examples to articulate principles that have guided computational biology broadly and call for renewed attention to these principles as computation becomes increasingly important in stem cell biology. We also discuss important challenges for this field with the hope that it will inspire more to join this exciting area.
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Affiliation(s)
- Patrick Cahan
- Institute for Cell Engineering, Department of Biomedical Engineering, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
| | - Davide Cacchiarelli
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy d Department of Translational Medicine, University of Naples "Federico II," Naples, Italy
| | - Sara-Jane Dunn
- DeepMind, 14-18 Handyside Street, London N1C 4DN, UK; Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre, Puddicombe Way, Cambridge Biomedical Campus, Cambridge CB2 0AW, UK
| | - Martin Hemberg
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK
| | | | - Samantha A Morris
- Department of Developmental Biology, Department of Genetics, Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Owen J L Rackham
- Centre for Computational Biology and The Program for Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore
| | - Antonio Del Sol
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 6 Avenue du Swing, Belvaux 4366, Luxembourg; CIC bioGUNE, Bizkaia Technology Park, 801 Building, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain
| | - Christine A Wells
- Centre for Stem Cell Systems, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, VIC 3010, Australia
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13
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McDonald D, Wu Y, Dailamy A, Tat J, Parekh U, Zhao D, Hu M, Tipps A, Zhang K, Mali P. Defining the Teratoma as a Model for Multi-lineage Human Development. Cell 2020; 183:1402-1419.e18. [PMID: 33152263 PMCID: PMC7704916 DOI: 10.1016/j.cell.2020.10.018] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2019] [Revised: 06/06/2020] [Accepted: 10/09/2020] [Indexed: 12/14/2022]
Abstract
We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.
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Affiliation(s)
- Daniella McDonald
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, San Diego, CA 92093, USA
| | - Yan Wu
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA
| | - Amir Dailamy
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA
| | - Justin Tat
- Department of Biological Sciences, University of California, San Diego, San Diego, CA 92093, USA
| | - Udit Parekh
- Department of Electrical and Computer Engineering, University of California, San Diego, San Diego, CA 92093, USA
| | - Dongxin Zhao
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA
| | - Michael Hu
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA
| | - Ann Tipps
- School of Medicine, University of California, San Diego, San Diego, CA 92103, USA
| | - Kun Zhang
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, San Diego, CA 92093, USA.
| | - Prashant Mali
- Department of Bioengineering, University of California, San Diego, San Diego, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, San Diego, CA 92093, USA.
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14
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Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation. Sci Rep 2020; 10:18582. [PMID: 33122739 PMCID: PMC7596514 DOI: 10.1038/s41598-020-75657-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2019] [Accepted: 10/15/2020] [Indexed: 01/01/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.
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15
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Panina Y, Karagiannis P, Kurtz A, Stacey GN, Fujibuchi W. Human Cell Atlas and cell-type authentication for regenerative medicine. Exp Mol Med 2020; 52:1443-1451. [PMID: 32929224 PMCID: PMC8080834 DOI: 10.1038/s12276-020-0421-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 03/05/2020] [Accepted: 03/09/2020] [Indexed: 12/22/2022] Open
Abstract
In modern biology, the correct identification of cell types is required for the developmental study of tissues and organs and the production of functional cells for cell therapies and disease modeling. For decades, cell types have been defined on the basis of morphological and physiological markers and, more recently, immunological markers and molecular properties. Recent advances in single-cell RNA sequencing have opened new doors for the characterization of cells at the individual and spatiotemporal levels on the basis of their RNA profiles, vastly transforming our understanding of cell types. The objective of this review is to survey the current progress in the field of cell-type identification, starting with the Human Cell Atlas project, which aims to sequence every cell in the human body, to molecular marker databases for individual cell types and other sources that address cell-type identification for regenerative medicine based on cell data guidelines.
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Affiliation(s)
- Yulia Panina
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Peter Karagiannis
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Andreas Kurtz
- BIH Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany
| | - Glyn N Stacey
- International Stem Cell Banking Initiative, 2 High Street, Barley, Herts, SG88HZ, UK
- National Stem Cell Resource Centre, Institute of Zoology, Chinese Academy of Sciences, 100190, Beijing, China
- Innovation Academy for Stem Cell and Regeneration, Chinese Academy of Sciences, 100101, Beijing, China
| | - Wataru Fujibuchi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
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16
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Park J, Lee NG, Oh M, Song J, Kim W, Kwon MG, Kim SG, Han BS, Bae KH, Lee DG, Lee SH, Park JG, Kim JH, Lee J, Min JK. Selective elimination of human pluripotent stem cells by Anti-Dsg2 antibody-doxorubicin conjugates. Biomaterials 2020; 259:120265. [PMID: 32827795 DOI: 10.1016/j.biomaterials.2020.120265] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2020] [Accepted: 07/26/2020] [Indexed: 01/19/2023]
Abstract
The self-renewal properties of human pluripotent stem cells (hPSCs) contribute to their efficacy in tissue regeneration applications yet increase the likelihood of teratoma formation, thereby limiting their clinical utility. To address this issue, we developed a tool to specifically target and neutralize undifferentiated hPSCs, thereby minimizing tumorigenicity risk without negatively affecting regenerated and somatic tissues. Specifically, we conjugated a monoclonal antibody (K6-1) previously generated in our laboratory against desmoglein 2 (Dsg2), which is highly differentially expressed in undifferentiated hPSCs versus somatic tissues, to the chemotherapeutic agent doxorubicin (DOX). The K6-1-DOX conjugates were selectively targeted and incorporated into Dsg2-positive hPSCs, leading to pH-dependent endosomal release and nuclear localization of DOX with subsequent cytotoxicity via an apoptotic caspase cascade. Conversely, Dsg2-negative fibroblasts showed minimal conjugate uptake or cytotoxicity, suggesting that K6-1-DOX treatment would yield few side effects owing to off-target effects. Selective removal of undifferentiated stem cells was also supported by in vivo studies using a mouse xenograft model, wherein hIgG-DOX- but not K6-1-DOX-pretreated-hPSC injection led to teratoma development. Together, these results validated the ability of the Dsg2-targeted antibody-anticancer drug conjugate to facilitate the safety of stem cell therapies.
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Affiliation(s)
- Jongjin Park
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Na Geum Lee
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
| | - Mihee Oh
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jinhoi Song
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Wooil Kim
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
| | - Min-Gi Kwon
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
| | - Seul Gi Kim
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
| | - Baek Soo Han
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Kwang-Hee Bae
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Dong Gwang Lee
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Sang-Hyun Lee
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jong-Gil Park
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jae Ho Kim
- Department of Physiology, Pusan National University Yangsan Hospital, Yangsan, 50612, Republic of Korea
| | - Jangwook Lee
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
| | - Jeong-Ki Min
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea.
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17
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Liew LC, Gailhouste L, Tan GC, Yamamoto Y, Takeshita F, Nakagama H, Ochiya T. MicroRNA-124a inhibits endoderm lineage commitment by targeting Sox17 and Gata6 in mouse embryonic stem cells. Stem Cells 2019; 38:504-515. [PMID: 31828873 PMCID: PMC7187259 DOI: 10.1002/stem.3136] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Accepted: 11/22/2019] [Indexed: 01/18/2023]
Abstract
The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, via miRNA profiling during endoderm differentiation, we discovered that miR‐124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). To further investigate the functional role of miR‐124a in early stages of differentiation, transfection of embryoid bodies with miR‐124a mimic was performed. We showed that overexpression of miR‐124a inhibits endoderm differentiation in vitro through targeting the 3′‐untranslated region (UTR) of Sox17 and Gata6, revealing the existence of interplay between miR‐124a and the Sox17/Gata6 transcription factors in hepato‐specific gene regulation. In addition, we presented a feasible in vivo system that utilizes teratoma and gene expression profiling from microarray to quantitatively evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR‐124a in teratomas by intratumor delivery of miR‐124a mimic and Atelocollagen, significantly suppressed endoderm and mesoderm lineage differentiation while augmenting the differentiation into ectoderm lineage. Collectively, our findings suggest that miR‐124a plays a significant role in mESCs lineage commitment.
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Affiliation(s)
- Lee Chuen Liew
- Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.,Department of Pathology, Immunology and Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Luc Gailhouste
- Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.,Liver Cancer Prevention Research Unit, RIKEN Cluster for Pioneering Research, Saitama, Japan
| | - Geok Chin Tan
- Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Yusuke Yamamoto
- Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan
| | - Fumitaka Takeshita
- Department of Functional Analysis, FIOC, National Cancer Center Research Institute, Tokyo, Japan
| | - Hitoshi Nakagama
- Department of Pathology, Immunology and Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.,National Cancer Center, Tokyo, Japan
| | - Takahiro Ochiya
- Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.,Department of Molecular and Cellular Medicine, Tokyo Medical University, Tokyo, Japan
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18
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Kim M, Kino‐oka M. Designing a blueprint for next‐generation stem cell bioprocessing development. Biotechnol Bioeng 2019; 117:832-843. [DOI: 10.1002/bit.27228] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2019] [Revised: 08/12/2019] [Accepted: 11/10/2019] [Indexed: 01/03/2023]
Affiliation(s)
- Mee‐Hae Kim
- Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuita Osaka Japan
| | - Masahiro Kino‐oka
- Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuita Osaka Japan
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19
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Liu W, Deng C, Godoy-Parejo C, Zhang Y, Chen G. Developments in cell culture systems for human pluripotent stem cells. World J Stem Cells 2019; 11:968-981. [PMID: 31768223 PMCID: PMC6851012 DOI: 10.4252/wjsc.v11.i11.968] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 08/21/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications.
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Affiliation(s)
- Weiwei Liu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Bioimaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Chunhao Deng
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Carlos Godoy-Parejo
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Yumeng Zhang
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Guokai Chen
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China.
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20
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Liu N, Yin Y, Wang H, Zhou Z, Sheng X, Fu H, Guo R, Wang H, Yang J, Gong P, Ning W, Ju Z, Liu Y, Liu L. Telomere dysfunction impairs epidermal stem cell specification and differentiation by disrupting BMP/pSmad/P63 signaling. PLoS Genet 2019; 15:e1008368. [PMID: 31518356 PMCID: PMC6760834 DOI: 10.1371/journal.pgen.1008368] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2019] [Revised: 09/25/2019] [Accepted: 08/12/2019] [Indexed: 11/19/2022] Open
Abstract
Telomere shortening is associated with aging and age-associated diseases. Additionally, telomere dysfunction resulting from telomerase gene mutation can lead to premature aging, such as apparent skin atrophy and hair loss. However, the molecular signaling linking telomere dysfunction to skin atrophy remains elusive. Here we show that dysfunctional telomere disrupts BMP/pSmad/P63 signaling, impairing epidermal stem cell specification and differentiation of skin and hair follicles. We find that telomere shortening mediated by Terc loss up-regulates Follistatin (Fst), inhibiting pSmad signaling and down-regulating P63 and epidermal keratins in an ESC differentiation model as well as in adult development of telomere-shortened mice. Mechanistically, short telomeres disrupt PRC2/H3K27me3-mediated repression of Fst. Our findings reveal that skin atrophy due to telomere dysfunction is caused by a previously unappreciated link with Fst and BMP signaling that could be explored in the development of therapies.
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Affiliation(s)
- Na Liu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
- Key Laboratory of Bioactive Materials, Ministry of Education, Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, China
- School of Medicine, Nankai University, Tianjin, China
| | - Yu Yin
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
- Yunnan Key Laboratory of Primate Biomedical Research; Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China
| | - Haiying Wang
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Zhongcheng Zhou
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Xiaoyan Sheng
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Haifeng Fu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Renpeng Guo
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Hua Wang
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Jiao Yang
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Peng Gong
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Wen Ning
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
| | - Zhenyu Ju
- Key Laboratory of Regenerative Medicine of Ministry of Education, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China
| | - Yifei Liu
- Yale Fertility Center and Department of OB/GYN, Yale University School of Medicine, New Haven, CT, United States of America
- * E-mail: (YL); (LL)
| | - Lin Liu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
- Key Laboratory of Bioactive Materials, Ministry of Education, Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, China
- * E-mail: (YL); (LL)
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21
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Liu LP, Zheng YW. Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges. World J Stem Cells 2019; 11:375-382. [PMID: 31396366 PMCID: PMC6682503 DOI: 10.4252/wjsc.v11.i7.375] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2019] [Revised: 06/12/2019] [Accepted: 06/20/2019] [Indexed: 02/06/2023] Open
Abstract
The capability of human pluripotent stem cell (hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by high-throughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo. Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation. For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost, restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.
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Affiliation(s)
- Li-Ping Liu
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Yun-Wen Zheng
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China.
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22
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Liu Y, Liu L, Zhou Y, Zhou P, Yan Q, Chen X, Ding S, Zhu F. CKLF1 Enhances Inflammation-Mediated Carcinogenesis and Prevents Doxorubicin-Induced Apoptosis via IL6/STAT3 Signaling in HCC. Clin Cancer Res 2019; 25:4141-4154. [PMID: 30918019 DOI: 10.1158/1078-0432.ccr-18-3510] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Revised: 02/24/2019] [Accepted: 03/20/2019] [Indexed: 12/21/2022]
Abstract
PURPOSE Hepatocellular carcinoma (HCC), one of the most common and deadliest malignancies worldwide, has a poor prognosis, owing to its high potential for vascular invasion and metastasis and the lack of biomarkers for early diagnosis. Thus, it must be a crucial factor for investigating therapeutic strategies for HCC to identify the functional molecular targets. Here, we reported a novel chemokine, CKLF1, that might act as a pivotal modulator in the invasion and metastasis of HCC and could serve as an attractive target for cancer therapy. EXPERIMENTAL DESIGN Bioinformatics analysis, PCR, Western blotting, and IHC were performed to detect the expression of CKLF1 in HCC. The function of CKLF1 was demonstrated by a series of in vitro and in vivo experiments. Pharmacologic treatment, flow cytometry, and Western blotting were carried out to demonstrate the potential mechanisms of CKLF1. RESULTS We proved that CKLF1 was overexpressed in HCC tissues and was related to tumor stage, vascular invasion, and patient survival. Then, functional assays showed that CKLF1 promoted hepatocellular carcinogenesis and metastatic potential. Finally, the IL6/STAT3 signaling pathway was involved in the mechanistic investigation. The results demonstrated that CKLF1 enhanced the progression of HCC and prevented doxorubicin-induced apoptosis through activating the IL6/STAT3 pathway. CONCLUSIONS These data showed that CKLF1 inhibited apoptosis and promoted malignant transformation through the IL6/STAT3 pathway, and ultimately enhanced the development and metastasis of HCC. Thus, our work revealed that CKLF1 was a significant prognostic factor of HCC and might be a potential molecular therapeutic target for HCC.
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Affiliation(s)
- Youyi Liu
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
- Department of Psychiatry, Renmin Hospital of Wuhan University, Wuhan, China
| | - Lijuan Liu
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
| | - Yan Zhou
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
| | - Ping Zhou
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
| | - Qiujin Yan
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
| | - Xiaobei Chen
- Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China
| | - Shuang Ding
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China
| | - Fan Zhu
- State Key Laboratory of Virology, Department of Medical Microbiology, School of Medicine, Wuhan University, Wuhan, China.
- Department of Psychiatry, Renmin Hospital of Wuhan University, Wuhan, China
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan, China
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23
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Georgomanoli M, Papapetrou EP. Modeling blood diseases with human induced pluripotent stem cells. Dis Model Mech 2019; 12:12/6/dmm039321. [PMID: 31171568 PMCID: PMC6602313 DOI: 10.1242/dmm.039321] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) are derived from somatic cells through a reprogramming process, which converts them to a pluripotent state, akin to that of embryonic stem cells. Over the past decade, iPSC models have found increasing applications in the study of human diseases, with blood disorders featuring prominently. Here, we discuss methodological aspects pertaining to iPSC generation, hematopoietic differentiation and gene editing, and provide an overview of uses of iPSCs in modeling the cell and gene therapy of inherited genetic blood disorders, as well as their more recent use as models of myeloid malignancies. We also discuss the strengths and limitations of iPSCs compared to model organisms and other cellular systems commonly used in hematology research.
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Affiliation(s)
- Maria Georgomanoli
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Eirini P Papapetrou
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
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24
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Addressing Variability and Heterogeneity of Induced Pluripotent Stem Cell-Derived Cardiomyocytes. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1212:1-29. [DOI: 10.1007/5584_2019_350] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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25
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Yoshida K, Okada M, Nagasaka R, Sasaki H, Okada M, Kanie K, Kato R. Time-course colony tracking analysis for evaluating induced pluripotent stem cell culture processes. J Biosci Bioeng 2019; 128:209-217. [PMID: 30738731 DOI: 10.1016/j.jbiosc.2019.01.011] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 01/11/2019] [Accepted: 01/16/2019] [Indexed: 01/15/2023]
Abstract
Increasing the yield and maintaining a high quality of induced pluripotent stem cells (iPSCs) is necessary for manufacturing iPSCs at the industrial scale. However, because iPSCs are delicate, it is important to evaluate their quality during processing. To examine the status of cultured iPSCs non-invasively, morphology-based iPSC colony evaluation may be an efficient technology for cellular status monitoring and analysis. In this study, we examined the effectiveness of time-course colony tracking analysis for evaluating the iPSC culture process. Particularly, we obtained detailed time-course data to evaluate the effect of the pipetting technique on cell dissociation before seeding. Although the pipetting process causes severe shear stress to cells, which affects their quality, these effects have not been quantitatively analyzed because of their complex and uncontrollable parameters. By analyzing the heterogeneity and time-course responses of individual colonies, our colony tracking analysis revealed a critically damaged population caused by pipetting stress which could not be detected in conventional bulk analysis. Moreover, by comprehensively analyzing colony tracking data, which links the time-course morphology and marker staining results with each colony, we found that colony morphology is only highly correlated with the undifferentiated marker in the final stage, with a lower correlation in the early stages. Thus, colony tracking analysis provides a way to quantify cellular morphological information when evaluating complex iPSC manufacturing processes.
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Affiliation(s)
- Kei Yoshida
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Mika Okada
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Risako Nagasaka
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Hiroto Sasaki
- Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Mai Okada
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Kei Kanie
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Ryuji Kato
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan; Stem Cell Evaluation Technology Research Association (SCA), Hacho-bori, Chuou-ku, Tokyo 104-0032, Japan; Institute of Nano-Life-Systems, Institute of Innovation for Future Society, Nagoya University, Division of Micro-Nano Mechatronics, Furocho, Chikusa-ku, Nagoya 464-8602, Japan.
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26
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Salvatori DCF, Dorssers LCJ, Gillis AJM, Perretta G, van Agthoven T, Gomes Fernandes M, Stoop H, Prins JB, Oosterhuis JW, Mummery C, Looijenga LHJ. The MicroRNA-371 Family as Plasma Biomarkers for Monitoring Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells in Teratoma Assays. Stem Cell Reports 2018; 11:1493-1505. [PMID: 30503260 PMCID: PMC6294243 DOI: 10.1016/j.stemcr.2018.11.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 11/01/2018] [Accepted: 11/01/2018] [Indexed: 01/09/2023] Open
Abstract
Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.
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Affiliation(s)
- Daniela C F Salvatori
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands.
| | - Lambert C J Dorssers
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Ad J M Gillis
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Gemma Perretta
- Fondazione Guido Bernardini, Via Manfredo Camperio, 10, 20123 Milano, Italy
| | - Ton van Agthoven
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Maria Gomes Fernandes
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands
| | - Hans Stoop
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Jan-Bas Prins
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands
| | - J Wolter Oosterhuis
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Christine Mummery
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Leendert H J Looijenga
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands.
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The International Stem Cell Initiative, Allison TF, Andrews PW, Avior Y, Barbaric I, Benvenisty N, Bock C, Brehm J, Brüstle O, Damjanov I, Elefanty A, Felkner D, Gokhale PJ, Halbritter F, Healy LE, Hu TX, Knowles BB, Loring JF, Ludwig TE, Mayberry R, Micallef S, Mohamed JS, Müller FJ, Mummery CL, Nakatsuji N, Ng ES, Oh SKW, O’Shea O, Pera MF, Reubinoff B, Robson P, Rossant J, Schuldt BM, Solter D, Sourris K, Stacey G, Stanley EG, Suemori H, Takahashi K, Yamanaka S. Assessment of established techniques to determine developmental and malignant potential of human pluripotent stem cells. Nat Commun 2018; 9:1925. [PMID: 29765017 PMCID: PMC5954055 DOI: 10.1038/s41467-018-04011-3] [Citation(s) in RCA: 77] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2017] [Accepted: 03/26/2018] [Indexed: 12/12/2022] Open
Abstract
The International Stem Cell Initiative compared several commonly used approaches to assess human pluripotent stem cells (PSC). PluriTest predicts pluripotency through bioinformatic analysis of the transcriptomes of undifferentiated cells, whereas, embryoid body (EB) formation in vitro and teratoma formation in vivo provide direct tests of differentiation. Here we report that EB assays, analyzed after differentiation under neutral conditions and under conditions promoting differentiation to ectoderm, mesoderm, or endoderm lineages, are sufficient to assess the differentiation potential of PSCs. However, teratoma analysis by histologic examination and by TeratoScore, which estimates differential gene expression in each tumor, not only measures differentiation but also allows insight into a PSC's malignant potential. Each of the assays can be used to predict pluripotent differentiation potential but, at this stage of assay development, only the teratoma assay provides an assessment of pluripotency and malignant potential, which are both relevant to the pre-clinical safety assessment of PSCs.
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28
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Bouma MJ, van Iterson M, Janssen B, Mummery CL, Salvatori DCF, Freund C. Differentiation-Defective Human Induced Pluripotent Stem Cells Reveal Strengths and Limitations of the Teratoma Assay and In Vitro Pluripotency Assays. Stem Cell Reports 2018; 8:1340-1353. [PMID: 28494940 PMCID: PMC5425621 DOI: 10.1016/j.stemcr.2017.03.009] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2016] [Revised: 03/10/2017] [Accepted: 03/10/2017] [Indexed: 02/06/2023] Open
Abstract
The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assays are best selected on the basis of intended downstream applications.
Side-by-side comparison of teratomas/TeratoScore, hPSC ScoreCard, and PluriTest hiPSCs with reactivated transgenes form embryonal carcinomas in vivo hiPSCs with reactivated transgenes show impaired differentiation capacity in vitro • PluriTest does not distinguish hiPSCs with reactivated transgenes from normal hPSCs
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Affiliation(s)
- Marga J Bouma
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Maarten van Iterson
- Department of Molecular Epidemiology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Bart Janssen
- GenomeScan B.V., Plesmanlaan 1D, 2333 BZ Leiden, the Netherlands
| | - Christine L Mummery
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Daniela C F Salvatori
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
| | - Christian Freund
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
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29
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Keller A, Dziedzicka D, Zambelli F, Markouli C, Sermon K, Spits C, Geens M. Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells. Hum Reprod Update 2018; 24:162-175. [PMID: 29377992 DOI: 10.1093/humupd/dmx042] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Revised: 11/23/2017] [Accepted: 12/22/2017] [Indexed: 01/19/2023] Open
Abstract
BACKGROUND Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. OBJECTIVE AND RATIONALE Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. SEARCH METHODS Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. OUTCOMES Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. WIDER IMPLICATIONS As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.
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Affiliation(s)
- Alexander Keller
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Dominika Dziedzicka
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Filippo Zambelli
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Christina Markouli
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Karen Sermon
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Claudia Spits
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Mieke Geens
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
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30
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31
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Secreto FJ, Li X, Smith AJ, Bruinsma ES, Perales-Clemente E, Oommen S, Hawse G, Hrstka SCL, Arendt BK, Brandt EB, Wigle DA, Nelson TJ. Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality. Stem Cells Transl Med 2017; 6:1829-1839. [PMID: 28924979 PMCID: PMC6430057 DOI: 10.1002/sctm.17-0116] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2017] [Accepted: 07/19/2017] [Indexed: 12/15/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high‐throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time‐consuming, and incompatible with high‐throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells’ documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419‐42‐0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine2017;6:1829–1839
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Affiliation(s)
- Frank J Secreto
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Xing Li
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota, USA
| | - Alyson J Smith
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Elizabeth S Bruinsma
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Ester Perales-Clemente
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Saji Oommen
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Gresin Hawse
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Sybil C L Hrstka
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Bonnie K Arendt
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Emma B Brandt
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Dennis A Wigle
- Division of Thoracic Surgery, Mayo Clinic, Rochester, Minnesota, USA.,Center for Regenerative Medicine BioTrust, Mayo Clinic, Rochester, Minnesota, USA
| | - Timothy J Nelson
- Program for Hypoplastic Left Heart Syndrome-Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Division of General Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Transplant Center, Mayo Clinic, Rochester, Minnesota, USA.,Division of Pediatric Cardiology, Mayo Clinic, Rochester, Minnesota, USA.,Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA.,Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA
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32
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Jeong HC, Park SJ, Choi JJ, Go YH, Hong SK, Kwon OS, Shin JG, Kim RK, Lee MO, Lee SJ, Shin HD, Moon SH, Cha HJ. PRMT8 Controls the Pluripotency and Mesodermal Fate of Human Embryonic Stem Cells By Enhancing the PI3K/AKT/SOX2 Axis. Stem Cells 2017; 35:2037-2049. [DOI: 10.1002/stem.2642] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 05/06/2017] [Indexed: 12/31/2022]
Affiliation(s)
- Ho-Chang Jeong
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Soon-Jung Park
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Jong-Jin Choi
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Young-Hyun Go
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Soon-Ki Hong
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Ok-Seon Kwon
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Joong-Gon Shin
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
- Research Institute for Basic Science, Sogang University; Seoul Republic of Korea
| | - Rae-Kwon Kim
- Department of Life Science; Research Institute for Natural Sciences, Hanyang University; Seoul Republic of Korea
| | - Mi-Ok Lee
- Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon Republic of Korea
| | - Su-Jae Lee
- Department of Life Science; Research Institute for Natural Sciences, Hanyang University; Seoul Republic of Korea
| | - Hyoung Doo Shin
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
- Research Institute for Basic Science, Sogang University; Seoul Republic of Korea
| | - Sung-Hwan Moon
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Hyuk-Jin Cha
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
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Radley AH, Schwab RM, Tan Y, Kim J, Lo EKW, Cahan P. Assessment of engineered cells using CellNet and RNA-seq. Nat Protoc 2017; 12:1089-1102. [PMID: 28448485 PMCID: PMC5765439 DOI: 10.1038/nprot.2017.022] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
CellNet is a computational platform designed to assess cell populations engineered by either directed differentiation of pluripotent stem cells (PSCs) or direct conversion, and to suggest specific hypotheses to improve cell fate engineering protocols. CellNet takes as input gene expression data and compares them with large data sets of normal expression profiles compiled from public sources, in regard to the extent to which cell- and tissue-specific gene regulatory networks are established. CellNet was originally designed to work with human or mouse microarray expression data for 21 cell or tissue (C/T) types. Here we describe how to apply CellNet to RNA-seq data and how to build a completely new CellNet platform applicable to, for example, other species or additional cell and tissue types. Once the raw data have been preprocessed, running CellNet takes only several minutes, whereas the time required to create a completely new CellNet is several hours.
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Affiliation(s)
- Arthur H Radley
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
| | - Remy M Schwab
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
| | - Yuqi Tan
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
| | - Jeesoo Kim
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
| | - Emily KW Lo
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
| | - Patrick Cahan
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA
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Avior Y, Lezmi E, Yanuka D, Benvenisty N. Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells. Stem Cell Reports 2017; 8:1354-1365. [PMID: 28392220 PMCID: PMC5425613 DOI: 10.1016/j.stemcr.2017.03.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2016] [Revised: 03/06/2017] [Accepted: 03/07/2017] [Indexed: 12/18/2022] Open
Abstract
Human embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1−/− hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1−/− cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1–/–-specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1.
RB1-null hESCs were generated using CRISPR/Cas9 RB1−/− hESCs generate large, neural-enriched teratomas, possibly by ZEB1 activation RB1 inactivation triggers aberrant mitochondrial abundance and function Unbiased drug screening found that carboplatin specifically targets RB1-null cells
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Affiliation(s)
- Yishai Avior
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Dorit Yanuka
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel.
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Bian Q, Cahan P. Computational Tools for Stem Cell Biology. Trends Biotechnol 2016; 34:993-1009. [PMID: 27318512 PMCID: PMC5116400 DOI: 10.1016/j.tibtech.2016.05.010] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2016] [Revised: 05/17/2016] [Accepted: 05/19/2016] [Indexed: 12/30/2022]
Abstract
For over half a century, the field of developmental biology has leveraged computation to explore mechanisms of developmental processes. More recently, computational approaches have been critical in the translation of high throughput data into knowledge of both developmental and stem cell biology. In the past several years, a new subdiscipline of computational stem cell biology has emerged that synthesizes the modeling of systems-level aspects of stem cells with high-throughput molecular data. In this review, we provide an overview of this new field and pay particular attention to the impact that single cell transcriptomics is expected to have on our understanding of development and our ability to engineer cell fate.
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Affiliation(s)
- Qin Bian
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Patrick Cahan
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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36
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Utilizing Regulatory Networks for Pluripotency Assessment in Stem Cells. CURRENT STEM CELL REPORTS 2016. [DOI: 10.1007/s40778-016-0054-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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Müller M, Hermann PC, Liebau S, Weidgang C, Seufferlein T, Kleger A, Perkhofer L. The role of pluripotency factors to drive stemness in gastrointestinal cancer. Stem Cell Res 2016; 16:349-57. [PMID: 26896855 DOI: 10.1016/j.scr.2016.02.005] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/08/2015] [Revised: 01/19/2016] [Accepted: 02/01/2016] [Indexed: 12/28/2022] Open
Abstract
A better molecular understanding of gastrointestinal cancers arising either from the stomach, the pancreas, the intestine, or the liver has led to the identification of a variety of potential new molecular therapeutic targets. However, in most cases surgery remains the only curative option. The intratumoral cellular heterogeneity of cancer stem cells, bulk tumor cells, and stromal cells further limits straightforward targeting approaches. Accumulating evidence reveals an intimate link between embryonic development, stem cells, and cancer formation. In line, a growing number of oncofetal proteins are found to play common roles within these processes. Cancer stem cells share features with true stem cells by having the capacity to self-renew in a de-differentiated state, to generate heterogeneous types of differentiated progeny, and to give rise to the bulk tumor. Further, various studies identified genes in cancer stem cells, which were previously shown to regulate the pluripotency circuitry, particularly the so-called "Yamanaka-Factors" (OCT4, KLF4, SOX2, and c-MYC). However, the true stemness potential of cancer stem cells and the role and expression pattern of such pluripotency genes in various tumor cell types remain to be explored. Here, we summarize recent findings and discuss the potential mechanisms involved, and link them to clinical significance with a particular focus on gastrointestinal cancers.
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Affiliation(s)
- Martin Müller
- Department of Internal Medicine I, Ulm University, Ulm, Germany
| | | | - Stefan Liebau
- Institute of Neuroanatomy, Eberhard Karls University Tuebingen, Tuebingen, Germany
| | - Clair Weidgang
- Department of Anesthesiology, Ulm University Hospital, Ulm, Germany
| | | | - Alexander Kleger
- Department of Internal Medicine I, Ulm University, Ulm, Germany.
| | - Lukas Perkhofer
- Department of Internal Medicine I, Ulm University, Ulm, Germany
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Park SJ, Komiyama Y, Suemori H, Umezawa A, Nakai K. OpenTein: a database of digital whole-slide images of stem cell-derived teratomas. Nucleic Acids Res 2016; 44:D1000-D1004. [PMID: 26496950 PMCID: PMC4702800 DOI: 10.1093/nar/gkv1096] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 10/07/2015] [Accepted: 10/11/2015] [Indexed: 12/01/2022] Open
Abstract
Human stem cells are promising sources for regenerative therapy. To ensure safety of future therapeutic applications, the differentiation potency of stem cells has to be tested and be widely opened to the public. The potency is generally assessed by teratoma formation comprising differentiated cells from all three germ layers, and the teratomas can be inspected through high-quality digital images. The teratoma assay, however, lacks consistency in transplantation protocols and even in interpretation, which needs community-based efforts for improving the assay quality. Here, we have developed a novel database OpenTein (Open Teratoma Investigation, http://opentein.hgc.jp/) to archive and freely distribute high-resolution whole-slide images and relevant records. OpenTein has been designed as a searchable, zoomable and annotatable web-based repository system. We have deposited 468 images of teratomas derived by our transplantation of human stem cells, and users can freely access and process such digital teratoma images. Approximately, the current version of OpenTein responds within 11.2 min for processing 2.03 gigapixel teratoma images. Our system offers valuable tools and resources in the new era of stem cell biology.
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Affiliation(s)
- Sung-Joon Park
- Human Genome Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
| | - Yusuke Komiyama
- Human Genome Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
| | - Hirofumi Suemori
- Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Akihiro Umezawa
- Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan
| | - Kenta Nakai
- Human Genome Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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