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Scuderi S, Kang TY, Jourdon A, Nelson A, Yang L, Wu F, Anderson GM, Mariani J, Tomasini L, Sarangi V, Abyzov A, Levchenko A, Vaccarino FM. Specification of human brain regions with orthogonal gradients of WNT and SHH in organoids reveals patterning variations across cell lines. Cell Stem Cell 2025; 32:970-989.e11. [PMID: 40315847 PMCID: PMC12145255 DOI: 10.1016/j.stem.2025.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 03/10/2025] [Accepted: 04/09/2025] [Indexed: 05/04/2025]
Abstract
The repertoire of neurons and their progenitors depends on their location along the antero-posterior and dorso-ventral axes of the neural tube. To model these axes, we designed the Dual Orthogonal-Morphogen Assisted Patterning System (Duo-MAPS) diffusion device to expose spheres of induced pluripotent stem cells (iPSCs) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison with single-cell transcriptomes from the fetal human brain revealed that Duo-MAPS-patterned organoids generated an extensive diversity of neuronal lineages from the forebrain, midbrain, and hindbrain. WNT and SHH crosstalk translated into early patterns of gene expression programs associated with the generation of specific brain lineages with distinct functional networks. Human iPSC lines showed substantial interindividual and line-to-line variations in their response to morphogens, highlighting that genetic and epigenetic variations may influence regional specification. Morphogen gradients promise to be a key approach to model the brain in its entirety.
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Affiliation(s)
- Soraya Scuderi
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | - Tae-Yun Kang
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Alexandre Jourdon
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | - Alex Nelson
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | - Liang Yang
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Feinan Wu
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | | | - Jessica Mariani
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | - Livia Tomasini
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA
| | - Vivekananda Sarangi
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA
| | - Alexej Abyzov
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA
| | - Andre Levchenko
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
| | - Flora M Vaccarino
- Program in Neurodevelopment and Regeneration, Yale University, New Haven, CT 06520, USA; Child Study Center, Yale University, New Haven, CT 06520, USA; Department of Neuroscience, Yale University, New Haven, CT 06520, USA; Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA.
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2
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Nie R, Zheng C, Ren L, Teng Y, Sun Y, Wang L, Li J, Cai J. Mitigating Cell Cycle Effects in Multi-Omics Data: Solutions and Analytical Frameworks. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025:e05823. [PMID: 40434003 DOI: 10.1002/advs.202505823] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/02/2025] [Revised: 04/30/2025] [Indexed: 05/29/2025]
Abstract
Cell cycle structures vary significantly across cell types, which exhibit distinct phase compositions. Asynchronous DNA replication and dynamic cellular characteristics during the cell cycle result in considerable heterogeneity in DNA dosage, chromatin accessibility, methylation, and expression. Nonetheless, the consequences of cell cycle disruption in the interpretation of multi-omics data remain unclear. Here, we systematically assessed the influence of distinct cell phase structures on the interpretation of omics features in proliferating cells, and proposed solutions for each omics dataset. For copy number variation (CNV) calling, asynchronous replication timing (RT) interference induces false CNVs in cells with high S-phase ratio (SPR), which are significantly decreased following replication timing domain (RTD) correction. Similar noise is observed in the chromatin accessibility data. Moreover, for DNA methylation and transcriptomic analyses, cell cycle-sorted data outperformed direct comparison in elucidating the biological features of compared cells. Additionally, we established an integrated pipeline to identify differentially expressed genes (DEGs) after cell cycle phasing. Consequently, our study demonstrated extensive cell-cycle heterogeneity, warranting consideration in future studies involving cells with diverse cell-cycle structures. RTD correction or phase-specific comparison could reduce the influence of cell cycle composition on the analysis of the differences observed between stem and differentiated cells.
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Affiliation(s)
- Rui Nie
- Chinese Academy of Sciences and China National Center for Bioinformation, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Caihong Zheng
- Laboratory of Molecular Biology and Bovine Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Likun Ren
- Chinese Academy of Sciences and China National Center for Bioinformation, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yue Teng
- Chinese Academy of Sciences and China National Center for Bioinformation, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yaoyu Sun
- School of Life Sciences, Peking University, Beijing, 100871, China
| | - Lifei Wang
- Department of Chemistry, the University of Hong Kong, Hong Kong, 999077, China
| | - Junya Li
- Laboratory of Molecular Biology and Bovine Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Jun Cai
- Chinese Academy of Sciences and China National Center for Bioinformation, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
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Pașca SP, Arlotta P, Bateup HS, Camp JG, Cappello S, Gage FH, Knoblich JA, Kriegstein AR, Lancaster MA, Ming GL, Novarino G, Okano H, Parmar M, Park IH, Reiner O, Song H, Studer L, Takahashi J, Temple S, Testa G, Treutlein B, Vaccarino FM, Vanderhaeghen P, Young-Pearse T. A framework for neural organoids, assembloids and transplantation studies. Nature 2025; 639:315-320. [PMID: 39653126 DOI: 10.1038/s41586-024-08487-6] [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: 08/26/2024] [Accepted: 12/04/2024] [Indexed: 02/20/2025]
Abstract
As the field of neural organoids and assembloids expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. In this Perspective, we present a framework for the experimental process that encompasses ensuring the quality and integrity of human pluripotent stem cells, characterizing and manipulating neural cells in vitro, transplantation techniques and considerations for modelling human development, evolution and disease. As with all scientific endeavours, we advocate for rigorous experimental designs tailored to explicit scientific questions as well as transparent methodologies and data sharing to provide useful knowledge for current research practices and for developing regulatory standards.
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Affiliation(s)
- Sergiu P Pașca
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.
- Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute and Bio-X, Stanford University, Stanford, CA, USA.
| | - Paola Arlotta
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Helen S Bateup
- Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA
- Department of Neuroscience, University of California Berkeley, Berkeley, CA, USA
| | - J Gray Camp
- Institute of Human Biology (IHB), Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
- Biozentrum, University of Basel, Basel, Switzerland
| | - Silvia Cappello
- Department of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig Maximilian University of Munich, Munich, Germany
- Max Planck Institute of Psychiatry, Munich, Germany
| | - Fred H Gage
- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA
| | - Jürgen A Knoblich
- Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna Biocenter, Vienna, Austria
- Department of Neurology, Medical University of Vienna, Vienna, Austria
| | - Arnold R Kriegstein
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA
- Department of Neurology, University of California San Francisco, San Francisco, CA, USA
| | - Madeline A Lancaster
- MRC Laboratory of Molecular Biology, Cambridge, UK
- Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Guo-Li Ming
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Cell and Developmental Biology, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Psychiatry, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Gaia Novarino
- Institute of Science and Technology of Austria, Klosterneuburg, Austria
| | - Hideyuki Okano
- Keio University Regenerative Medicine Research Center, Kanagawa, Japan
| | - Malin Parmar
- Department of Experimental Medical Science, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - In-Hyun Park
- Department of Genetics, Yale School of Medicine, New Haven, CT, USA
- Yale Stem Cell Center, Yale University, New Haven, CT, USA
| | - Orly Reiner
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
- Department of Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel
| | - Hongjun Song
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Cell and Developmental Biology, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
- The Epigenetics Institute, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA
| | - Jun Takahashi
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | | | - Giuseppe Testa
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
- Human Technopole, Viale Rita Levi Montalcini, Milan, Italy
| | - Barbara Treutlein
- Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
| | - Flora M Vaccarino
- Yale Stem Cell Center, Yale University, New Haven, CT, USA
- Child Study Center, Yale University, New Haven, CT, USA
- Department of Neuroscience, Yale University, New Haven, CT, USA
- Yale Kavli Institute for Neuroscience, New Haven, CT, USA
| | - Pierre Vanderhaeghen
- VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
- Department of Neurosciences, Leuven Brain Institute, Leuven, Belgium
| | - Tracy Young-Pearse
- Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA
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Zhou W, Mumm C, Gan Y, Switzenberg JA, Wang J, De Oliveira P, Kathuria K, Losh SJ, McDonald TL, Bessell B, Van Deynze K, McConnell MJ, Boyle AP, Mills RE. A personalized multi-platform assessment of somatic mosaicism in the human frontal cortex. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.18.629274. [PMID: 39763954 PMCID: PMC11702624 DOI: 10.1101/2024.12.18.629274] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/16/2025]
Abstract
Somatic mutations in individual cells lead to genomic mosaicism, contributing to the intricate regulatory landscape of genetic disorders and cancers. To evaluate and refine the detection of somatic mosaicism across different technologies with personalized donor-specific assembly (DSA), we obtained tissue from the dorsolateral prefrontal cortex (DLPFC) of a post-mortem neurotypical 31-year-old individual. We sequenced bulk DLPFC tissue using Oxford Nanopore Technologies (~60X), NovaSeq (~30X), and linked-read sequencing (~28X). Additionally, we applied Cas9 capture methodology coupled with long-read sequencing (TEnCATS), targeting active transposable elements. We also isolated and amplified DNA from flow-sorted single DLPFC neurons using MALBAC, sequencing 115 of these MALBAC libraries on Nanopore and 94 on NovaSeq. We constructed a haplotype-resolved assembly with a total length of 5.77 Gb and a phase block length of 2.67 Mb (N50) to facilitate cross-platform analysis of somatic genetic variations. We observed an increase in the phasing rate from 11.6% to 38.0% between short-read and long-read technologies. By generating a catalog of phased germline SNVs, CNVs, and TEs from the assembled genome, we applied standard approaches to recall these variants across sequencing technologies. We achieved aggregated recall rates from 97.3% to 99.4% based on long-read bulk tissue data, setting an upper bound for detection limits. Moreover, utilizing haplotype-based analysis from DSA, we achieved a remarkable reduction in false positive somatic calls in bulk tissue, ranging from 14.9% to 72.4%. We developed pipelines leveraging DSA information to enhance somatic large genetic variant calling in long-read single cells. By examining somatic variation using long-reads in 115 individual neurons, we identified 468 candidate somatic heterozygous large deletions (1.5Mb - 20Mb), 137 of which intersected with short-read single-cell data. Additionally, we identified 61 putative somatic TEs (60 Alus, one LINE-1) in the single-cell data. Collectively, our analysis spans personalized assembly to single-cell somatic variant calling, providing a comprehensive ab initio ad finem approach and resource in real human tissue.
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Affiliation(s)
- Weichen Zhou
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Camille Mumm
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
| | - Yanming Gan
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Jessica A. Switzenberg
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Jinhao Wang
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | | | - Kunal Kathuria
- Lieber Institute for Brain Development, Baltimore, MD, USA
| | - Steven J. Losh
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Torrin L. McDonald
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Brandt Bessell
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | - Kinsey Van Deynze
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
| | | | - Alan P. Boyle
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
| | - Ryan E. Mills
- Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
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5
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Williams MJ, Oliphant MUJ, Au V, Liu C, Baril C, O'Flanagan C, Lai D, Beatty S, Van Vliet M, Yiu JC, O'Connor L, Goh WL, Pollaci A, Weiner AC, Grewal D, McPherson A, Norton K, Moore M, Prabhakar V, Agarwal S, Garber JE, Dillon DA, Shah SP, Brugge JS, Aparicio S. Luminal breast epithelial cells of BRCA1 or BRCA2 mutation carriers and noncarriers harbor common breast cancer copy number alterations. Nat Genet 2024; 56:2753-2762. [PMID: 39567747 PMCID: PMC11631757 DOI: 10.1038/s41588-024-01988-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Accepted: 10/15/2024] [Indexed: 11/22/2024]
Abstract
The prevalence and nature of somatic copy number alterations (CNAs) in breast epithelium and their role in tumor initiation and evolution remain poorly understood. Using single-cell DNA sequencing (49,238 cells) of epithelium from BRCA1 and BRCA2 carriers or wild-type individuals, we identified recurrent CNAs (for example, 1q-gain and 7q, 10q, 16q and 22q-loss) that are present in a rare population of cells across almost all samples (n = 28). In BRCA1/BRCA2 carriers, these occur before loss of heterozygosity (LOH) of wild-type alleles. These CNAs, common in malignant tumors, are enriched in luminal cells but absent in basal myoepithelial cells. Allele-specific analysis of prevalent CNAs reveals that they arose by independent mutational events, consistent with convergent evolution. BRCA1/BRCA2 carriers contained a small percentage of cells with extreme aneuploidy, featuring loss of TP53, BRCA1/BRCA2 LOH and multiple breast cancer-associated CNAs. Our findings suggest that CNAs arising in normal luminal breast epithelium are precursors to clonally expanded tumor genomes.
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Affiliation(s)
- Marc J Williams
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
- The Halvorsen Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
| | - Michael U J Oliphant
- Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School (HMS), Boston, MA, USA
| | - Vinci Au
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Cathy Liu
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Caroline Baril
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Ciara O'Flanagan
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Daniel Lai
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Sean Beatty
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Michael Van Vliet
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Jacky Ch Yiu
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Lauren O'Connor
- Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School (HMS), Boston, MA, USA
| | - Walter L Goh
- Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School (HMS), Boston, MA, USA
| | - Alicia Pollaci
- Department of Medical Oncology, Dana-Farber Cancer Institute (DFCI), Boston, MA, USA
| | - Adam C Weiner
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
- The Halvorsen Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
| | - Diljot Grewal
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
- The Halvorsen Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
| | - Andrew McPherson
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
- The Halvorsen Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
| | - Klarisa Norton
- Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School (HMS), Boston, MA, USA
| | - McKenna Moore
- Department of Medical Oncology, Dana-Farber Cancer Institute (DFCI), Boston, MA, USA
| | - Vikas Prabhakar
- Department of Pathology, Brigham and Women's Hospital (BWH), Boston, MA, USA
| | - Shailesh Agarwal
- Department of Surgery, Brigham and Women's Hospital (BWH), Boston, MA, USA
| | - Judy E Garber
- Department of Medical Oncology, Dana-Farber Cancer Institute (DFCI), Boston, MA, USA
| | - Deborah A Dillon
- Department of Medical Oncology, Dana-Farber Cancer Institute (DFCI), Boston, MA, USA
| | - Sohrab P Shah
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA.
- The Halvorsen Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York City, NY, USA.
| | - Joan S Brugge
- Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School (HMS), Boston, MA, USA.
| | - Samuel Aparicio
- Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada.
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
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Vijayraghavan S, Blouin T, McCollum J, Porcher L, Virard F, Zavadil J, Feghali-Bostwick C, Saini N. Widespread mutagenesis and chromosomal instability shape somatic genomes in systemic sclerosis. Nat Commun 2024; 15:8889. [PMID: 39406724 PMCID: PMC11480385 DOI: 10.1038/s41467-024-53332-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2024] [Accepted: 10/09/2024] [Indexed: 10/19/2024] Open
Abstract
Systemic sclerosis is a connective tissue disorder characterized by excessive fibrosis that primarily affects women, and can present as a multisystem pathology. Roughly 4-22% of patients with systemic sclerosis develop cancer, which drastically worsens prognosis. However, the mechanisms underlying systemic sclerosis initiation, propagation, and cancer development are poorly understood. We hypothesize that the inflammation and immune response associated with systemic sclerosis can trigger DNA damage, leading to elevated somatic mutagenesis, a hallmark of pre-cancerous tissues. To test our hypothesis, we culture clonal lineages of fibroblasts from the lung tissues of controls and systemic sclerosis patients and compare their mutation burdens and spectra. We find an overall increase in all major mutation types in systemic sclerosis samples compared to control lung samples, from small-scale events such as single base substitutions and insertions/deletions, to chromosome-level changes, including copy-number changes and structural variants. In the genomes of patients with systemic sclerosis, we find evidence of somatic hypermutation or kategis (typically only seen in cancer genomes), we identify mutation signatures closely resembling the error-prone translesion polymerase Polη activity, and observe an activation-induced deaminase-like mutation signature, which overlaps with genomic regions displaying kataegis.
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Affiliation(s)
- Sriram Vijayraghavan
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA
| | - Thomas Blouin
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA
| | - James McCollum
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA
| | - Latarsha Porcher
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA
| | - François Virard
- University Claude Bernard Lyon 1, INSERM U1052-CNRS UMR5286, Cancer Research Center, Centre Léon Bérard, Lyon, France
| | - Jiri Zavadil
- International Agency for Research on Cancer WHO, Epigenomics and Mechanisms Branch, Lyon, France
| | - Carol Feghali-Bostwick
- Department of Medicine, Division of Rheumatology, Medical University of South Carolina, Charleston, SC, USA
| | - Natalie Saini
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.
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7
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Sol S, Boncimino F, Todorova K, Mandinova A. Unraveling the Functional Heterogeneity of Human Skin at Single-Cell Resolution. Hematol Oncol Clin North Am 2024; 38:921-938. [PMID: 38839486 DOI: 10.1016/j.hoc.2024.05.001] [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: 06/07/2024]
Abstract
The skin consists of several cell populations, including epithelial, immune, and stromal cells. Recently, there has been a significant increase in single-cell RNA-sequencing studies, contributing to the development of a consensus Human Skin Cell Atlas. The aim is to understand skin biology better and identify potential therapeutic targets. The present review utilized previously published single-cell RNA-sequencing datasets to explore human skin's cellular and functional heterogeneity. Additionally, it summarizes the functional significance of newly identified cell subpopulations in processes such as wound healing and aging.
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Affiliation(s)
- Stefano Sol
- Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Fabiana Boncimino
- Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Kristina Todorova
- Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Anna Mandinova
- Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Broad Institute of Harvard and MIT, 7 Cambridge Center, MA 02142, USA; Harvard Stem Cell Institute, 7 Divinity Avenue Cambridge, MA 02138, USA.
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8
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Riew TR, Kim YS. Mutational Landscapes of Normal Skin and Their Potential Implications in the Development of Skin Cancer: A Comprehensive Narrative Review. J Clin Med 2024; 13:4815. [PMID: 39200957 PMCID: PMC11355262 DOI: 10.3390/jcm13164815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2024] [Revised: 08/11/2024] [Accepted: 08/13/2024] [Indexed: 09/02/2024] Open
Abstract
Recent evidence suggests that physiologically normal skin harbors pervasive mutant clones with cancer drivers. Normal skin has the highest burden of somatic mutations due to persistent ultraviolet exposure throughout life. The mutation burden exponentially increases with age and is further modified by skin site, sun-damage history, and skin phototype. Driver gene profiles in normal skin are similar to those in cutaneous squamous cell carcinoma where NOTCH family, FAT family, and TP53 are consistently reported, while other reported profiles include PPM1D, KMT2D, ASXL1, and RBM10. Normal skin seldom harbors canonical hotspot mutations with therapeutic relevance. The pathologic role of mutant clones with cancer drivers in normal skin is classically considered precursors for skin cancer; however, recent evidence also suggests their putative cancer-protective role. Copy number alterations and other structural variants are rare in normal skin with loss in 9q region encompassing NOTCH1 being the most common. Study methodologies should be carefully designed to obtain an adequate number of cells for sequencing, and a comparable number of cells and read depth across samples. In conclusion, this review provides mutational landscapes of normal skin and discusses their potential implications in the development of skin cancer, highlighting the role of driver genes in early malignant progression.
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Affiliation(s)
- Tae-Ryong Riew
- Department of Anatomy, Catholic Neuroscience Institute, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
- Department of Biomedicine and Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Yoon-Seob Kim
- Department of Dermatology, Bucheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
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9
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Park TY, Jeon J, Cha Y, Kim KS. Past, present, and future of cell replacement therapy for parkinson's disease: a novel emphasis on host immune responses. Cell Res 2024; 34:479-492. [PMID: 38777859 PMCID: PMC11217403 DOI: 10.1038/s41422-024-00971-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Accepted: 04/28/2024] [Indexed: 05/25/2024] Open
Abstract
Parkinson's disease (PD) stands as the second most common neurodegenerative disorder after Alzheimer's disease, and its prevalence continues to rise with the aging global population. Central to the pathophysiology of PD is the specific degeneration of midbrain dopamine neurons (mDANs) in the substantia nigra. Consequently, cell replacement therapy (CRT) has emerged as a promising treatment approach, initially supported by various open-label clinical studies employing fetal ventral mesencephalic (fVM) cells. Despite the initial favorable results, fVM cell therapy has intrinsic and logistical limitations that hinder its transition to a standard treatment for PD. Recent efforts in the field of cell therapy have shifted its focus towards the utilization of human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, to surmount existing challenges. However, regardless of the transplantable cell sources (e.g., xenogeneic, allogeneic, or autologous), the poor and variable survival of implanted dopamine cells remains a major obstacle. Emerging evidence highlights the pivotal role of host immune responses following transplantation in influencing the survival of implanted mDANs, underscoring an important area for further research. In this comprehensive review, building upon insights derived from previous fVM transplantation studies, we delve into the functional ramifications of host immune responses on the survival and efficacy of grafted dopamine cells. Furthermore, we explore potential strategic approaches to modulate the host immune response, ultimately aiming for optimal outcomes in future clinical applications of CRT for PD.
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Affiliation(s)
- Tae-Yoon Park
- Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, Belmont, MA, USA
- Program in Neuroscience, Harvard Medical School, Belmont, MA, USA
| | - Jeha Jeon
- Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, Belmont, MA, USA
- Program in Neuroscience, Harvard Medical School, Belmont, MA, USA
| | - Young Cha
- Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, Belmont, MA, USA
- Program in Neuroscience, Harvard Medical School, Belmont, MA, USA
| | - Kwang-Soo Kim
- Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, Belmont, MA, USA.
- Program in Neuroscience, Harvard Medical School, Belmont, MA, USA.
- Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
- Harvard Stem Cell Institute, Harvard Medical School, Belmont, MA, USA.
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10
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Graham JH, Schlachetzki JCM, Yang X, Breuss MW. Genomic Mosaicism of the Brain: Origin, Impact, and Utility. Neurosci Bull 2024; 40:759-776. [PMID: 37898991 PMCID: PMC11178748 DOI: 10.1007/s12264-023-01124-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Accepted: 07/16/2023] [Indexed: 10/31/2023] Open
Abstract
Genomic mosaicism describes the phenomenon where some but not all cells within a tissue harbor unique genetic mutations. Traditionally, research focused on the impact of genomic mosaicism on clinical phenotype-motivated by its involvement in cancers and overgrowth syndromes. More recently, we increasingly shifted towards the plethora of neutral mosaic variants that can act as recorders of cellular lineage and environmental exposures. Here, we summarize the current state of the field of genomic mosaicism research with a special emphasis on our current understanding of this phenomenon in brain development and homeostasis. Although the field of genomic mosaicism has a rich history, technological advances in the last decade have changed our approaches and greatly improved our knowledge. We will provide current definitions and an overview of contemporary detection approaches for genomic mosaicism. Finally, we will discuss the impact and utility of genomic mosaicism.
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Affiliation(s)
- Jared H Graham
- Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado School of Medicine, Aurora, 80045-2581, CO, USA
| | - Johannes C M Schlachetzki
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, 92093-0021, San Diego, CA, USA
| | - Xiaoxu Yang
- Department of Neurosciences, University of California San Diego, La Jolla, 92093-0021, San Diego, CA, USA
- Rady Children's Institute for Genomic Medicine, San Diego, 92123, CA, USA
| | - Martin W Breuss
- Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado School of Medicine, Aurora, 80045-2581, CO, USA.
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11
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Williams MJ, Oliphant MU, Au V, Liu C, Baril C, O'Flanagan C, Lai D, Beatty S, Van Vliet M, Yiu JC, O'Connor L, Goh WL, Pollaci A, Weiner AC, Grewal D, McPherson A, Moore M, Prabhakar V, Agarwal S, Garber JE, Dillon D, Shah SP, Brugge J, Aparicio S. Luminal breast epithelial cells from wildtype and BRCA mutation carriers harbor copy number alterations commonly associated with breast cancer. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.01.591587. [PMID: 38746396 PMCID: PMC11092623 DOI: 10.1101/2024.05.01.591587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of TP53 , LOH of BRCA1 or BRCA2 , and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.
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12
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Goecke T, Ius F, Ruhparwar A, Martin U. Unlocking the Future: Pluripotent Stem Cell-Based Lung Repair. Cells 2024; 13:635. [PMID: 38607074 PMCID: PMC11012168 DOI: 10.3390/cells13070635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 03/18/2024] [Accepted: 03/26/2024] [Indexed: 04/13/2024] Open
Abstract
The human respiratory system is susceptible to a variety of diseases, ranging from chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis to acute respiratory distress syndrome (ARDS). Today, lung diseases represent one of the major challenges to the health care sector and represent one of the leading causes of death worldwide. Current treatment options often focus on managing symptoms rather than addressing the underlying cause of the disease. The limitations of conventional therapies highlight the urgent clinical need for innovative solutions capable of repairing damaged lung tissue at a fundamental level. Pluripotent stem cell technologies have now reached clinical maturity and hold immense potential to revolutionize the landscape of lung repair and regenerative medicine. Meanwhile, human embryonic (HESCs) and human-induced pluripotent stem cells (hiPSCs) can be coaxed to differentiate into lung-specific cell types such as bronchial and alveolar epithelial cells, or pulmonary endothelial cells. This holds the promise of regenerating damaged lung tissue and restoring normal respiratory function. While methods for targeted genetic engineering of hPSCs and lung cell differentiation have substantially advanced, the required GMP-grade clinical-scale production technologies as well as the development of suitable preclinical animal models and cell application strategies are less advanced. This review provides an overview of current perspectives on PSC-based therapies for lung repair, explores key advances, and envisions future directions in this dynamic field.
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Affiliation(s)
- Tobias Goecke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs, Lower Saxony Center for Biomedical Engineering, Implant Research and Development /Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; (F.I.); (A.R.)
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
- Biomedical Research in End-stage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | - Fabio Ius
- Leibniz Research Laboratories for Biotechnology and Artificial Organs, Lower Saxony Center for Biomedical Engineering, Implant Research and Development /Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; (F.I.); (A.R.)
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
- Biomedical Research in End-stage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | - Arjang Ruhparwar
- Leibniz Research Laboratories for Biotechnology and Artificial Organs, Lower Saxony Center for Biomedical Engineering, Implant Research and Development /Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; (F.I.); (A.R.)
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
- Biomedical Research in End-stage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | - Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs, Lower Saxony Center for Biomedical Engineering, Implant Research and Development /Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; (F.I.); (A.R.)
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
- Biomedical Research in End-stage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
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13
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Kapadia CD, Goodell MA. Tissue mosaicism following stem cell aging: blood as an exemplar. NATURE AGING 2024; 4:295-308. [PMID: 38438628 DOI: 10.1038/s43587-024-00589-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Accepted: 02/07/2024] [Indexed: 03/06/2024]
Abstract
Loss of stem cell regenerative potential underlies aging of all tissues. Somatic mosaicism, the emergence of cellular patchworks within tissues, increases with age and has been observed in every organ yet examined. In the hematopoietic system, as in most tissues, stem cell aging through a variety of mechanisms occurs in lockstep with the emergence of somatic mosaicism. Here, we draw on insights from aging hematopoiesis to illustrate fundamental principles of stem cell aging and somatic mosaicism. We describe the generalizable changes intrinsic to aged stem cells and their milieu that provide the backdrop for somatic mosaicism to emerge. We discuss genetic and nongenetic mechanisms that can result in tissue somatic mosaicism and existing methodologies to detect such clonal outgrowths. Finally, we propose potential avenues to modify mosaicism during aging, with the ultimate aim of increasing tissue resiliency.
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Affiliation(s)
- Chiraag D Kapadia
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
- Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA
| | - Margaret A Goodell
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
- Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA.
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14
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Xue Y, Su Z, Lin X, Ho MK, Yu KHO. Single-cell lineage tracing with endogenous markers. Biophys Rev 2024; 16:125-139. [PMID: 38495438 PMCID: PMC10937880 DOI: 10.1007/s12551-024-01179-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Accepted: 01/18/2024] [Indexed: 03/19/2024] Open
Abstract
Resolving lineage relationships between cells in an organism provides key insights into the fate of individual cells and drives a fundamental understanding of the process of development and disease. A recent rapid increase in experimental and computational advances for detecting naturally occurring somatic nuclear and mitochondrial mutation at single-cell resolution has expanded lineage tracing from model organisms to humans. This review discusses the advantages and challenges of experimental and computational techniques for cell lineage tracing using somatic mutation as endogenous DNA barcodes to decipher the relationships between cells during development and tumour evolution. We outlook the advantages of spatial clonal evolution analysis and single-cell lineage tracing using endogenous genetic markers.
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Affiliation(s)
- Yan Xue
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
- Laboratory of Data Discovery for Health Limited (D24H), Hong Kong Science Park, Units 1201-1206, 1223 & 1225, 12/F, Building 19W, 19 Science Park West Avenue, Hong Kong Science Park, Pak Shek Kok, New Territories, Hong Kong SAR, China
| | - Zezhuo Su
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
- Laboratory of Data Discovery for Health Limited (D24H), Hong Kong Science Park, Units 1201-1206, 1223 & 1225, 12/F, Building 19W, 19 Science Park West Avenue, Hong Kong Science Park, Pak Shek Kok, New Territories, Hong Kong SAR, China
- Department of Orthopaedics and Traumatology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | - Xinyi Lin
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | - Mun Kay Ho
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | - Ken H. O. Yu
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
- Laboratory of Data Discovery for Health Limited (D24H), Hong Kong Science Park, Units 1201-1206, 1223 & 1225, 12/F, Building 19W, 19 Science Park West Avenue, Hong Kong Science Park, Pak Shek Kok, New Territories, Hong Kong SAR, China
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15
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Roemen GMJM, Theunissen TEJ, Hoezen WWJ, Steyls ARM, Paulussen ADC, Mosterd K, Rahikkala E, zur Hausen A, Speel EJM, van Geel M. Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome. Biomedicines 2024; 12:330. [PMID: 38397932 PMCID: PMC10886644 DOI: 10.3390/biomedicines12020330] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Revised: 01/20/2024] [Accepted: 01/24/2024] [Indexed: 02/25/2024] Open
Abstract
Basal cell nevus syndrome (BCNS) is an inherited disorder characterized mainly by the development of basal cell carcinomas (BCCs) at an early age. BCNS is caused by heterozygous small-nucleotide variants (SNVs) and copy-number variants (CNVs) in the Patched1 (PTCH1) gene. Genetic diagnosis may be complicated in mosaic BCNS patients, as accurate SNV and CNV analysis requires high-sensitivity methods due to possible low variant allele frequencies. We compared test outcomes for PTCH1 CNV detection using multiplex ligation-probe amplification (MLPA) and digital droplet PCR (ddPCR) with samples from a BCNS patient heterozygous for a PTCH1 CNV duplication and the patient's father, suspected to have a mosaic form of BCNS. ddPCR detected a significantly increased PTCH1 copy-number ratio in the index patient's blood, and the father's blood and tissues, indicating that the father was postzygotic mosaic and the index patient inherited the CNV from him. MLPA only detected the PTCH1 duplication in the index patient's blood and in hair and saliva from the mosaic father. Our data indicate that ddPCR more accurately detects CNVs, even in low-grade mosaic BCNS patients, which may be missed by MLPA. In general, quantitative ddPCR can be of added value in the genetic diagnosis of mosaic BCNS patients and in estimating the recurrence risk for offspring.
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Affiliation(s)
- Guido M. J. M. Roemen
- Department of Pathology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands; (T.E.J.T.)
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
| | - Tom E. J. Theunissen
- Department of Pathology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands; (T.E.J.T.)
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
| | - Ward W. J. Hoezen
- Department of Dermatology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
| | - Anja R. M. Steyls
- Department of Clinical Genetics, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands;
| | - Aimee D. C. Paulussen
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
- Department of Clinical Genetics, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands;
| | - Klara Mosterd
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
- Department of Dermatology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
| | - Elisa Rahikkala
- Research Unit of Clinical Medicine, Department of Clinical Genetics, Medical Research Center Oulu, Oulu University Hospital, University of Oulu, 90570 Oulu, Finland
| | - Axel zur Hausen
- Department of Pathology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands; (T.E.J.T.)
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
| | - Ernst Jan M. Speel
- Department of Pathology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands; (T.E.J.T.)
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
| | - Michel van Geel
- GROW School for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands; (A.D.C.P.)
- Department of Dermatology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
- Department of Clinical Genetics, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands;
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16
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Edwards N, Combrinck C, McCaughey-Chapman A, Connor B. Directly reprogrammed fragile X syndrome dorsal forebrain precursor cells generate cortical neurons exhibiting impaired neuronal maturation. Front Cell Neurosci 2023; 17:1254412. [PMID: 37810261 PMCID: PMC10552551 DOI: 10.3389/fncel.2023.1254412] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Accepted: 09/01/2023] [Indexed: 10/10/2023] Open
Abstract
Introduction The neurodevelopmental disorder fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability associated with autism spectrum disorder. Inaccessibility to developing human brain cells is a major barrier to studying FXS. Direct-to-neural precursor reprogramming provides a unique platform to investigate the developmental profile of FXS-associated phenotypes throughout neural precursor and neuron generation, at a temporal resolution not afforded by post-mortem tissue and in a patient-specific context not represented in rodent models. Direct reprogramming also circumvents the protracted culture times and low efficiency of current induced pluripotent stem cell strategies. Methods We have developed a chemically modified mRNA (cmRNA) -based direct reprogramming protocol to generate dorsal forebrain precursors (hiDFPs) from FXS patient-derived fibroblasts, with subsequent differentiation to glutamatergic cortical neurons and astrocytes. Results We observed differential expression of mature neuronal markers suggesting impaired neuronal development and maturation in FXS- hiDFP-derived neurons compared to controls. FXS- hiDFP-derived cortical neurons exhibited dendritic growth and arborization deficits characterized by reduced neurite length and branching consistent with impaired neuronal maturation. Furthermore, FXS- hiDFP-derived neurons exhibited a significant decrease in the density of pre- and post- synaptic proteins and reduced glutamate-induced calcium activity, suggesting impaired excitatory synapse development and functional maturation. We also observed a reduced yield of FXS- hiDFP-derived neurons with a significant increase in FXS-affected astrocytes. Discussion This study represents the first reported derivation of FXS-affected cortical neurons following direct reprogramming of patient fibroblasts to dorsal forebrain precursors and subsequently neurons that recapitulate the key molecular hallmarks of FXS as it occurs in human tissue. We propose that direct to hiDFP reprogramming provides a unique platform for further study into the pathogenesis of FXS as well as the identification and screening of new drug targets for the treatment of FXS.
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Affiliation(s)
| | | | | | - Bronwen Connor
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
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17
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Jourdon A, Wu F, Mariani J, Capauto D, Norton S, Tomasini L, Amiri A, Suvakov M, Schreiner JD, Jang Y, Panda A, Nguyen CK, Cummings EM, Han G, Powell K, Szekely A, McPartland JC, Pelphrey K, Chawarska K, Ventola P, Abyzov A, Vaccarino FM. Modeling idiopathic autism in forebrain organoids reveals an imbalance of excitatory cortical neuron subtypes during early neurogenesis. Nat Neurosci 2023; 26:1505-1515. [PMID: 37563294 PMCID: PMC10573709 DOI: 10.1038/s41593-023-01399-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Accepted: 06/30/2023] [Indexed: 08/12/2023]
Abstract
Idiopathic autism spectrum disorder (ASD) is highly heterogeneous, and it remains unclear how convergent biological processes in affected individuals may give rise to symptoms. Here, using cortical organoids and single-cell transcriptomics, we modeled alterations in the forebrain development between boys with idiopathic ASD and their unaffected fathers in 13 families. Transcriptomic changes suggest that ASD pathogenesis in macrocephalic and normocephalic probands involves an opposite disruption of the balance between excitatory neurons of the dorsal cortical plate and other lineages such as early-generated neurons from the putative preplate. The imbalance stemmed from divergent expression of transcription factors driving cell fate during early cortical development. While we did not find genomic variants in probands that explained the observed transcriptomic alterations, a significant overlap between altered transcripts and reported ASD risk genes affected by rare variants suggests a degree of gene convergence between rare forms of ASD and the developmental transcriptome in idiopathic ASD.
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Affiliation(s)
- Alexandre Jourdon
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Feinan Wu
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Jessica Mariani
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Davide Capauto
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Scott Norton
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Livia Tomasini
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Anahita Amiri
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Milovan Suvakov
- Department of Quantitative Health Sciences, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA
| | - Jeremy D Schreiner
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Yeongjun Jang
- Department of Quantitative Health Sciences, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA
| | - Arijit Panda
- Department of Quantitative Health Sciences, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA
| | - Cindy Khanh Nguyen
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Elise M Cummings
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Gloria Han
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Kelly Powell
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Anna Szekely
- Department of Neurology, Yale University School of Medicine, New Haven, CT, USA
| | - James C McPartland
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Kevin Pelphrey
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
- Brain Institute, Department of Neurology, University of Virginia School of Medicine, Charlottesville, VA, USA
| | | | - Pamela Ventola
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA
| | - Alexej Abyzov
- Department of Quantitative Health Sciences, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA.
| | - Flora M Vaccarino
- Child Study Center, Yale University School of Medicine, New Haven, CT, USA.
- Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA.
- Kavli Institute for Neuroscience, Yale University, New Haven, CT, USA.
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Andrawus M, Sharvit L, Touitou N, Lerrer B, Cohen HY, Atzmon G. Copy number variation as a tool for implementing pregnancy as an aging model. Aging (Albany NY) 2023; 15:7922-7932. [PMID: 37639552 PMCID: PMC10496986 DOI: 10.18632/aging.204936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 07/10/2023] [Indexed: 08/31/2023]
Abstract
Copy number variations (CNV) are a major contributor to genome variability and have been linked to aging and other degradable phenotypes such as pregnancy physiology. To demonstrate how pregnancy can be used as a model of aging, we used CNVs from pregnant mice. Candidate CNVs were selected by applying case-control analysis in human centenarians compared with control groups. These CNVs were aligned with the mouse genome and their copy variation was assessed using qRT-PCR in liver and blood tissue samples from pregnant mice throughout pregnancy (baseline; first, second, and third trimester; post-partum). Eight of the ten selected CNVs demonstrated a significant decline/increase trend throughout the pregnancy followed by opposite direction soon after delivery in the liver and blood of the mouse tissues. Furthermore, significant differential expression was detected among the candidate CNVs' close vicinity genes (APA2A, LSS, RBDHF1, PLAAT1, and SCL17A2), but not in the WSCD2 gene. Establishing a genetic link between longevity and pregnancy is a significant step toward implementing the pregnancy process as a model for aging. These results in pregnant mice highlight the mechanism and similarities between pregnancy and aging. Investigating the mechanisms that cause such rejuvenation after labor could change our aging treatment paradigm.
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Affiliation(s)
- Mariana Andrawus
- Department of Human Biology, University of Haifa, Haifa 3498838, Israel
| | - Lital Sharvit
- Department of Human Biology, University of Haifa, Haifa 3498838, Israel
| | - Noga Touitou
- Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
| | - Batia Lerrer
- Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
| | - Haim Y. Cohen
- Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
| | - Gil Atzmon
- Department of Human Biology, University of Haifa, Haifa 3498838, Israel
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19
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Ali HRW, Suliman S, Osman TAH, Carrasco M, Bruland O, Costea DE, Ræder H, Mustafa K. Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues. Stem Cell Res Ther 2023; 14:199. [PMID: 37559144 PMCID: PMC10410907 DOI: 10.1186/s13287-023-03403-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 06/15/2023] [Indexed: 08/11/2023] Open
Abstract
BACKGROUND Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness. METHODS iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods. RESULTS More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective "whitish" outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type. CONCLUSIONS The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.
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Affiliation(s)
- Hassan R W Ali
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Salwa Suliman
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Tarig Al-Hadi Osman
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Manuel Carrasco
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
- Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway
| | - Ove Bruland
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Daniela-Elena Costea
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
- Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway
- Gade Laboratory for Pathology, Haukeland University Hospital, Bergen, Norway
| | - Helge Ræder
- Department of Clinical Science, University of Bergen, Bergen, Norway.
- Department of Pediatrics, Haukeland University Hospital, Bergen, Norway.
| | - Kamal Mustafa
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway.
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20
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Identification of marker genes to monitor residual iPSCs in iPSC-derived products. Cytotherapy 2023; 25:59-67. [PMID: 36319564 DOI: 10.1016/j.jcyt.2022.09.010] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 09/08/2022] [Accepted: 09/27/2022] [Indexed: 12/27/2022]
Abstract
BACKGROUND Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types. METHODS Identifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A). RESULTS Embryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used. CONCLUSIONS Using the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.
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21
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Gerdes P, Lim SM, Ewing AD, Larcombe MR, Chan D, Sanchez-Luque FJ, Walker L, Carleton AL, James C, Knaupp AS, Carreira PE, Nefzger CM, Lister R, Richardson SR, Polo JM, Faulkner GJ. Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells. Nat Commun 2022; 13:7470. [PMID: 36463236 PMCID: PMC9719517 DOI: 10.1038/s41467-022-35180-x] [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: 02/09/2022] [Accepted: 11/22/2022] [Indexed: 12/04/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
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Affiliation(s)
- Patricia Gerdes
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Sue Mei Lim
- grid.1002.30000 0004 1936 7857Department of Anatomy & Developmental Biology, Monash University, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800 Australia
| | - Adam D. Ewing
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Michael R. Larcombe
- grid.1002.30000 0004 1936 7857Department of Anatomy & Developmental Biology, Monash University, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800 Australia
| | - Dorothy Chan
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Francisco J. Sanchez-Luque
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia ,grid.418805.00000 0004 0500 8423GENYO. Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncological Research, PTS, Granada, 18016 Spain
| | - Lucinda Walker
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Alexander L. Carleton
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Cini James
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Anja S. Knaupp
- grid.1002.30000 0004 1936 7857Department of Anatomy & Developmental Biology, Monash University, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800 Australia
| | - Patricia E. Carreira
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Christian M. Nefzger
- grid.1002.30000 0004 1936 7857Department of Anatomy & Developmental Biology, Monash University, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800 Australia
| | - Ryan Lister
- grid.1012.20000 0004 1936 7910Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA 6009 Australia ,grid.431595.f0000 0004 0469 0045Harry Perkins Institute of Medical Research, Perth, WA 6009 Australia
| | - Sandra R. Richardson
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia
| | - Jose M. Polo
- grid.1002.30000 0004 1936 7857Department of Anatomy & Developmental Biology, Monash University, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800 Australia ,grid.1002.30000 0004 1936 7857Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800 Australia ,grid.1010.00000 0004 1936 7304Adelaide Centre for Epigenetics and The South Australian Immunogenomics Cancer Institute, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA 5005 Australia
| | - Geoffrey J. Faulkner
- grid.1003.20000 0000 9320 7537Mater Research Institute - University of Queensland, TRI Building, Woolloongabba, QLD 4102 Australia ,grid.1003.20000 0000 9320 7537Queensland Brain Institute, University of Queensland, Brisbane, QLD 4072 Australia
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22
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Dvir E, Shohat S, Flint J, Shifman S. Identification of genetic mechanisms for tissue-specific genetic effects based on CRISPR screens. Genetics 2022; 222:iyac134. [PMID: 36063051 PMCID: PMC9630981 DOI: 10.1093/genetics/iyac134] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2022] [Accepted: 08/26/2022] [Indexed: 11/12/2022] Open
Abstract
A major challenge in genetic studies of complex diseases is to determine how the action of risk genes is restricted to a tissue or cell type. Here, we investigate tissue specificity of gene action using CRISPR screens from 786 cancer cell lines originating from 24 tissues. We find that the expression pattern of the gene across tissues explains only a minority of cases of tissue-specificity (9%), while gene amplification and the expression levels of paralogs account for 39.5% and 15.5%, respectively. In addition, the transfer of small molecules to mutant cells explains tissue-specific gene action in blood. The tissue-specific genes we found are not specific just for human cancer cell lines: we found that the tissue-specific genes are intolerant to functional mutations in the human population and are associated with human diseases more than genes that are essential across all cell types. Our findings offer important insights into genetic mechanisms for tissue specificity of human diseases.
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Affiliation(s)
- Elad Dvir
- Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
| | - Shahar Shohat
- Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
| | - Jonathan Flint
- Department of Psychiatry and Biobehavioral Sciences, Brain Research Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Sagiv Shifman
- Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
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23
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Mohiuddin M, Kooy RF, Pearson CE. De novo mutations, genetic mosaicism and human disease. Front Genet 2022; 13:983668. [PMID: 36226191 PMCID: PMC9550265 DOI: 10.3389/fgene.2022.983668] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Accepted: 09/08/2022] [Indexed: 11/23/2022] Open
Abstract
Mosaicism—the existence of genetically distinct populations of cells in a particular organism—is an important cause of genetic disease. Mosaicism can appear as de novo DNA mutations, epigenetic alterations of DNA, and chromosomal abnormalities. Neurodevelopmental or neuropsychiatric diseases, including autism—often arise by de novo mutations that usually not present in either of the parents. De novo mutations might occur as early as in the parental germline, during embryonic, fetal development, and/or post-natally, through ageing and life. Mutation timing could lead to mutation burden of less than heterozygosity to approaching homozygosity. Developmental timing of somatic mutation attainment will affect the mutation load and distribution throughout the body. In this review, we discuss the timing of de novo mutations, spanning from mutations in the germ lineage (all ages), to post-zygotic, embryonic, fetal, and post-natal events, through aging to death. These factors can determine the tissue specific distribution and load of de novo mutations, which can affect disease. The disease threshold burden of somatic de novo mutations of a particular gene in any tissue will be important to define.
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Affiliation(s)
- Mohiuddin Mohiuddin
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
- *Correspondence: Mohiuddin Mohiuddin, ; Christopher E. Pearson,
| | - R. Frank Kooy
- Department of Medical Genetics, University of Antwerp, Edegem, Belgium
| | - Christopher E. Pearson
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
- *Correspondence: Mohiuddin Mohiuddin, ; Christopher E. Pearson,
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24
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Rouhani FJ, Zou X, Danecek P, Badja C, Amarante TD, Koh G, Wu Q, Memari Y, Durbin R, Martincorena I, Bassett AR, Gaffney D, Nik-Zainal S. Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nat Genet 2022; 54:1406-1416. [PMID: 35953586 PMCID: PMC9470532 DOI: 10.1038/s41588-022-01147-3] [Citation(s) in RCA: 51] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 06/24/2022] [Indexed: 12/27/2022]
Abstract
We explored human induced pluripotent stem cells (hiPSCs) derived from different tissues to gain insights into genomic integrity at single-nucleotide resolution. We used genome sequencing data from two large hiPSC repositories involving 696 hiPSCs and daughter subclones. We find ultraviolet light (UV)-related damage in ~72% of skin fibroblast-derived hiPSCs (F-hiPSCs), occasionally resulting in substantial mutagenesis (up to 15 mutations per megabase). We demonstrate remarkable genomic heterogeneity between independent F-hiPSC clones derived during the same round of reprogramming due to oligoclonal fibroblast populations. In contrast, blood-derived hiPSCs (B-hiPSCs) had fewer mutations and no UV damage but a high prevalence of acquired BCOR mutations (26.9% of lines). We reveal strong selection pressure for BCOR mutations in F-hiPSCs and B-hiPSCs and provide evidence that they arise in vitro. Directed differentiation of hiPSCs and RNA sequencing showed that BCOR mutations have functional consequences. Our work strongly suggests that detailed nucleotide-resolution characterization is essential before using hiPSCs.
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Affiliation(s)
- Foad J Rouhani
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Xueqing Zou
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK
- Academic Department of Medical Genetics, Addenbrooke's Treatment Centre, Cambridge Biomedical Research Campus, Cambridge, UK
| | - Petr Danecek
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Cherif Badja
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK
- Academic Department of Medical Genetics, Addenbrooke's Treatment Centre, Cambridge Biomedical Research Campus, Cambridge, UK
| | - Tauanne Dias Amarante
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK
| | - Gene Koh
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK
- Academic Department of Medical Genetics, Addenbrooke's Treatment Centre, Cambridge Biomedical Research Campus, Cambridge, UK
| | - Qianxin Wu
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Yasin Memari
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK
| | - Richard Durbin
- Department of Genetics, University of Cambridge, Cambridge, UK
| | - Inigo Martincorena
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Andrew R Bassett
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Daniel Gaffney
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
- Genomics plc, King Charles House, Oxford, UK
| | - Serena Nik-Zainal
- Early Cancer Institute, Hutchison/MRC Research Centre, Cambridge Biomedical Research Campus, Cambridge, UK.
- Academic Department of Medical Genetics, Addenbrooke's Treatment Centre, Cambridge Biomedical Research Campus, Cambridge, UK.
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25
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The effects of sequencing depth on the assembly of coding and noncoding transcripts in the human genome. BMC Genomics 2022; 23:487. [PMID: 35787153 PMCID: PMC9251931 DOI: 10.1186/s12864-022-08717-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Accepted: 06/16/2022] [Indexed: 12/30/2022] Open
Abstract
Investigating the functions and activities of genes requires proper annotation of the transcribed units. However, transcript assembly efforts have produced a surprisingly large variation in the number of transcripts, and especially so for noncoding transcripts. This heterogeneity in assembled transcript sets might be partially explained by sequencing depth. Here, we used real and simulated short-read sequencing data as well as long-read data to systematically investigate the impact of sequencing depths on the accuracy of assembled transcripts. We assembled and analyzed transcripts from 671 human short-read data sets and four long-read data sets. At the first level, there is a positive correlation between the number of reads and the number of recovered transcripts. However, the effect of the sequencing depth varied based on cell or tissue type, the type of read and the nature and expression levels of the transcripts. The detection of coding transcripts saturated rapidly with both short and long-reads, however, there was no sign of early saturation for noncoding transcripts at any sequencing depth. Increasing long-read sequencing depth specifically benefited transcripts containing transposable elements. Finally, we show how single-cell RNA-seq can be guided by transcripts assembled from bulk long-read samples, and demonstrate that noncoding transcripts are expressed at similar levels to coding transcripts but are expressed in fewer cells. This study highlights the impact of sequencing depth on transcript assembly.
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26
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Quist E, Trovato F, Avaliani N, Zetterdahl OG, Gonzalez-Ramos A, Hansen MG, Kokaia M, Canals I, Ahlenius H. Transcription factor-based direct conversion of human fibroblasts to functional astrocytes. Stem Cell Reports 2022; 17:1620-1635. [PMID: 35750047 PMCID: PMC9287681 DOI: 10.1016/j.stemcr.2022.05.015] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 05/20/2022] [Accepted: 05/23/2022] [Indexed: 12/14/2022] Open
Abstract
Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.
Effective direct conversion of human fibroblasts to induced astrocytes (iAs) iAs resemble human primary astrocytes at molecular, phenotypic, and functional levels iAs can be generated from fibroblasts covering the entire human lifespan iAs support function of induced neurons obtained from the same starting population
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Affiliation(s)
- Ella Quist
- Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Stem Cells, Aging and Neurodegeneration, Lund, Sweden; Lund Stem Cell Center, Lund, Sweden.
| | - Francesco Trovato
- Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Stem Cells, Aging and Neurodegeneration, Lund, Sweden; Lund Stem Cell Center, Lund, Sweden; Lund University, Skane University Hospital, Department of Clinical Sciences Lund, Neurosurgery, Lund, Sweden
| | | | - Oskar G Zetterdahl
- Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Stem Cells, Aging and Neurodegeneration, Lund, Sweden; Lund Stem Cell Center, Lund, Sweden; Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Glial and Neuronal Biology, Lund, Sweden
| | - Ana Gonzalez-Ramos
- Lund University, Skane University Hospital, Department of Clinical Sciences Lund, Epilepsy Center, Lund, Sweden
| | | | - Merab Kokaia
- Lund University, Skane University Hospital, Department of Clinical Sciences Lund, Epilepsy Center, Lund, Sweden
| | - Isaac Canals
- Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Glial and Neuronal Biology, Lund, Sweden
| | - Henrik Ahlenius
- Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Stem Cells, Aging and Neurodegeneration, Lund, Sweden; Lund Stem Cell Center, Lund, Sweden.
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27
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Tanaka T, Shiba T, Honda Y, Izawa K, Yasumi T, Saito MK, Nishikomori R. Induced Pluripotent Stem Cell-Derived Monocytes/Macrophages in Autoinflammatory Diseases. Front Immunol 2022; 13:870535. [PMID: 35603217 PMCID: PMC9120581 DOI: 10.3389/fimmu.2022.870535] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 04/11/2022] [Indexed: 11/13/2022] Open
Abstract
The concept of autoinflammation, first proposed in 1999, refers to a seemingly unprovoked episode of sterile inflammation manifesting as unexplained fever, skin rashes, and arthralgia. Autoinflammatory diseases are caused mainly by hereditary abnormalities of innate immunity, without the production of autoantibodies or autoreactive T cells. The revolutionary discovery of induced pluripotent stem cells (iPSCs), whereby a patient’s somatic cells can be reprogrammed into an embryonic pluripotent state by forced expression of a defined set of transcription factors, has the transformative potential to enable in vitro disease modeling and drug candidate screening, as well as to provide a resource for cell replacement therapy. Recent reports demonstrate that recapitulating a disease phenotype in vitro is feasible for numerous monogenic diseases, including autoinflammatory diseases. In this review, we provide a comprehensive overview of current advances in research into autoinflammatory diseases involving iPSC-derived monocytes/macrophages. This review may aid in the planning of new studies of autoinflammatory diseases.
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Affiliation(s)
- Takayuki Tanaka
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
- Department of Pediatrics, Japanese Red Cross Otsu Hospital, Otsu, Japan
- *Correspondence: Takayuki Tanaka,
| | - Takeshi Shiba
- Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, INSERM UMR 1163, Imagine Institute, Paris, France
| | - Yoshitaka Honda
- Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan
- Department of Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kazushi Izawa
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Takahiro Yasumi
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Megumu K. Saito
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Ryuta Nishikomori
- Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan
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Tian P, Elefanty A, Stanley EG, Durnall JC, Thompson LH, Elwood NJ. Creation of GMP-Compliant iPSCs From Banked Umbilical Cord Blood. Front Cell Dev Biol 2022; 10:835321. [PMID: 35372371 PMCID: PMC8967326 DOI: 10.3389/fcell.2022.835321] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Accepted: 02/15/2022] [Indexed: 12/24/2022] Open
Abstract
Many clinical trials are in progress using cells derived from induced pluripotent stem cells (iPSC) for immunotherapies and regenerative medicine. The success of these new therapies is underpinned by the quality of the cell population used to create the iPSC lines, along with the creation of iPSCs in a fully Good Manufacturing Practice (GMP)-compliant environment such that they can be used safely and effectively in the clinical setting. Umbilical cord blood (CB) from public cord blood banks is an excellent source of starting material for creation of iPSCs. All CB units are manufactured under GMP-conditions, have been screened for infectious diseases, with known family and medical history of the donor. Furthermore, the HLA tissue typing is known, thereby allowing identification of CB units with homozygous HLA haplotypes. CB cells are naïve with less exposure to environmental insults and iPSC can be generated with high efficiency. We describe a protocol that can be adopted by those seeking to create clinical-grade iPSC from banked CB. This protocol uses a small volume of thawed CB buffy to first undergo ex-vivo expansion towards erythroid progenitor cells, which are then used for reprogramming using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Resultant iPSC lines are tested to confirm pluripotency, genomic integrity, and stability. Cells are maintained in a feeder-free, xeno-free environment, using fully defined, commercially available reagents. Adoption of this protocol, with heed given to tips provided, allows efficient and robust creation of clinical-grade iPSC cell lines from small volumes of cryopreserved CB.
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Affiliation(s)
- Pei Tian
- Blood Development, Murdoch Children’s Research Institute, Melbourne, VIC, Australia
| | - Andrew Elefanty
- Blood Development, Murdoch Children’s Research Institute, Melbourne, VIC, Australia
- Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia
| | - Edouard G. Stanley
- Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia
- Immune Development, Murdoch Children’s Research Institute, Melbourne, VIC, Australia
| | - Jennifer C. Durnall
- Florey Institute of Neuroscience and Mental Health, Melbourne, VIC, Australia
| | - Lachlan H. Thompson
- Florey Institute of Neuroscience and Mental Health, Melbourne, VIC, Australia
| | - Ngaire J. Elwood
- Blood Development, Murdoch Children’s Research Institute, Melbourne, VIC, Australia
- Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia
- BMDI Cord Blood Bank, Melbourne, VIC, Australia
- *Correspondence: Ngaire J. Elwood,
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29
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Poetsch MS, Strano A, Guan K. Human induced pluripotent stem cells: From cell origin, genomic stability and epigenetic memory to translational medicine. Stem Cells 2022; 40:546-555. [PMID: 35291013 PMCID: PMC9216482 DOI: 10.1093/stmcls/sxac020] [Citation(s) in RCA: 60] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 03/06/2022] [Indexed: 11/14/2022]
Abstract
The potential of human induced pluripotent stem cells (iPSCs) to self-renew indefinitely and to differentiate virtually into any cell type in unlimited quantities makes them attractive for in-vitro disease modeling, drug screening, personalized medicine, and regenerative therapies. As the genome of iPSCs thoroughly reproduces that of the somatic cells from which they are derived, they may possess genetic abnormalities, which would seriously compromise their utility and safety. Genetic aberrations could be present in donor somatic cells and then transferred during iPSC generation, or they could occur as de novo mutations during reprogramming or prolonged cell culture. Therefore, to warrant safety of human iPSCs for clinical applications, analysis of genetic integrity, particularly during iPSC generation and differentiation, should be carried out on a regular basis. On the other hand, reprogramming of somatic cells to iPSCs requires profound modifications in the epigenetic landscape. Changes in chromatin structure by DNA methylations and histone tail modifications aim to reset the gene expression pattern of somatic cells to facilitate and establish self-renewal and pluripotency. However, residual epigenetic memory influences the iPSC phenotype, which may affect their application in disease therapeutics. The present review discusses the somatic cell origin, genetic stability, and epigenetic memory of iPSCs and their impact on basic and translational research.
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Affiliation(s)
- Mareike S Poetsch
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
| | - Anna Strano
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
| | - Kaomei Guan
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
- Corresponding author: Kaomei Guan, Institute of Pharmacology and Toxicology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany. Tel: +49 351 458 6246; Fax: +49 351 458 6315;
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Deb BK, Bateup HS. Modeling Somatic Mutations Associated With Neurodevelopmental Disorders in Human Brain Organoids. Front Mol Neurosci 2022; 14:787243. [PMID: 35058746 PMCID: PMC8764387 DOI: 10.3389/fnmol.2021.787243] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Accepted: 11/30/2021] [Indexed: 11/13/2022] Open
Abstract
Neurodevelopmental disorders (NDDs) are a collection of diseases with early life onset that often present with developmental delay, cognitive deficits, and behavioral conditions. In some cases, severe outcomes such as brain malformations and intractable epilepsy can occur. The mutations underlying NDDs may be inherited or de novo, can be gain- or loss-of-function, and can affect one or more genes. Recent evidence indicates that brain somatic mutations contribute to several NDDs, in particular malformations of cortical development. While advances in sequencing technologies have enabled the detection of these somatic mutations, the mechanisms by which they alter brain development and function are not well understood due to limited model systems that recapitulate these events. Human brain organoids have emerged as powerful models to study the early developmental events of the human brain. Brain organoids capture the developmental progression of the human brain and contain human-enriched progenitor cell types. Advances in human stem cell and genome engineering provide an opportunity to model NDD-associated somatic mutations in brain organoids. These organoids can be tracked throughout development to understand the impact of somatic mutations on early human brain development and function. In this review, we discuss recent evidence that somatic mutations occur in the developing human brain, that they can lead to NDDs, and discuss how they could be modeled using human brain organoids.
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Affiliation(s)
- Bipan K. Deb
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
| | - Helen S. Bateup
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
- Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, United States
- Chan Zuckerberg Biohub, San Francisco, CA, United States
- *Correspondence: Helen S. Bateup
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31
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Keller A, Spits C. The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells. Cells 2021; 10:cells10113246. [PMID: 34831467 PMCID: PMC8625075 DOI: 10.3390/cells10113246] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 11/07/2021] [Accepted: 11/17/2021] [Indexed: 12/23/2022] Open
Abstract
Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.
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Choudhury S, Surendran N, Das A. Recent advances in the induced pluripotent stem cell-based skin regeneration. Wound Repair Regen 2021; 29:697-710. [PMID: 33970525 DOI: 10.1111/wrr.12925] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 03/30/2021] [Accepted: 04/27/2021] [Indexed: 01/05/2023]
Abstract
Skin regeneration has been a challenging clinical problem especially in cases of chronic wounds such as diabetic foot ulcers, and epidermolysis bullosa-related skin blisters. Prolonged non-healing wounds often lead to bacterial infections increasing the severity of wounds. Current treatment strategies for chronic wounds include debridement of wounds along with antibiotics, growth factors, and stem cell transplantation therapies. However, the compromised nature of autologous stem cells in patients with comorbidities such as diabetes limits the efficacy of the therapy. The discovery of induced pluripotent stem cell (iPSC) technology has immensely influenced the field of regenerative therapy. Enormous efforts have been made to develop integration-free iPSCs suitable for clinical therapies. This review focuses on recent advances in the methods and reprogramming factors for generating iPSCs along with the existing challenges such as genetic alterations, tumorigenicity, immune rejection, and regulatory hurdles for the clinical application of iPSCs. Furthermore, this review also highlights the benefits of using iPSCs for the generation of skin cells and skin disease modeling over the existing clinical therapies for skin regeneration in chronic wounds and skin diseases.
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Affiliation(s)
- Subholakshmi Choudhury
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
- Academy of Science and Innovative Research (AcSIR), Ghaziabad, India
| | - Nidhi Surendran
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
| | - Amitava Das
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
- Academy of Science and Innovative Research (AcSIR), Ghaziabad, India
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33
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Somatic Mutations and Autoimmunity. Cells 2021; 10:cells10082056. [PMID: 34440825 PMCID: PMC8394445 DOI: 10.3390/cells10082056] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 08/04/2021] [Accepted: 08/05/2021] [Indexed: 11/17/2022] Open
Abstract
Autoimmune diseases are among the most common chronic illness caused by a dysregulated immune response against self-antigens. Close to 5% of the general population in Western countries develops some form of autoimmunity, yet its underlying causes, although intensively studied, are still not fully known, and no curative therapies exist. It is well established that autoimmune diseases have common mechanisms and are caused by both genetic and non-genetic risk factors. One novel risk factor that can contribute to autoimmunity is somatic mutations, in a role parallel to their role in cancer. Somatic mutations are stochastic, de novo, non-inherited mutations. In this hypothesis, the persistent proliferation of self-reactive lymphocytes (that is usually hindered by a series of checkpoints) is permitted, due to somatic mutations in these expanding cells, allowing them to bypass multiple regulatory checkpoints, causing autoimmunity. This novel concept of the contribution of these mutations in non-malignant diseases has recently started to be explored. It proposes a novel paradigm for autoimmunity etiology and could be the missing piece of the autoimmunity puzzle.
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34
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Cai YM, Dudley QM, Patron NJ. Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR. Bio Protoc 2021; 11:e4075. [PMID: 34327272 PMCID: PMC8292117 DOI: 10.21769/bioprotoc.4075] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Revised: 03/25/2021] [Accepted: 03/30/2021] [Indexed: 11/02/2022] Open
Abstract
Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability. Graphic abstract: Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.
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Affiliation(s)
- Yao-Min Cai
- Earlham Institute, Norwich Research Park, Colney lane, Norwich, UK
| | | | - Nicola J. Patron
- Earlham Institute, Norwich Research Park, Colney lane, Norwich, UK
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35
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Olafsson S, Anderson CA. Somatic mutations provide important and unique insights into the biology of complex diseases. Trends Genet 2021; 37:872-881. [PMID: 34226062 DOI: 10.1016/j.tig.2021.06.012] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 06/15/2021] [Accepted: 06/16/2021] [Indexed: 10/20/2022]
Abstract
Somatic evolution of cells within the body is well known to lead to cancers. However, spread of somatic mutations within a tissue over time may also contribute to the pathogenesis of non-neoplastic diseases. Recent years have seen the publication of many studies aiming to characterize somatic evolution in healthy tissues. A logical next step is to extend such work to diseased conditions. As our understanding of the interplay between somatic mutations and non-neoplastic disease grows, opportunities for the joint study of germline and somatic variants will present themselves. Here, we present our thoughts on the utility of somatic mutations for understanding both the causes and consequences of common complex disease and the challenges that remain for the joint study of the soma and germline.
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Affiliation(s)
| | - Carl A Anderson
- Wellcome Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK.
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36
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Zhang L, Pu K, Liu X, Bae SDW, Nguyen R, Bai S, Li Y, Qiao L. The Application of Induced Pluripotent Stem Cells Against Liver Diseases: An Update and a Review. Front Med (Lausanne) 2021; 8:644594. [PMID: 34277651 PMCID: PMC8280311 DOI: 10.3389/fmed.2021.644594] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Accepted: 06/04/2021] [Indexed: 11/13/2022] Open
Abstract
Liver diseases are a major health concern globally, and are associated with poor survival and prognosis of patients. This creates the need for patients to accept the main alternative treatment of liver transplantation to prevent progression to end-stage liver disease. Investigation of the molecular mechanisms underpinning complex liver diseases and their pathology is an emerging goal of stem cell scope. Human induced pluripotent stem cells (hiPSCs) derived from somatic cells are a promising alternative approach to the treatment of liver disease, and a prospective model for studying complex liver diseases. Here, we review hiPSC technology of cell reprogramming and differentiation, and discuss the potential application of hiPSC-derived liver cells, such as hepatocytes and cholangiocytes, in refractory liver-disease modeling and treatment, and drug screening and toxicity testing. We also consider hiPSC safety in clinical applications, based on genomic and epigenetic alterations, tumorigenicity, and immunogenicity.
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Affiliation(s)
- Lei Zhang
- The First Clinical Medical College, Lanzhou University, Lanzhou, China
- Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory of Biological Therapy and Regenerative Medicine Transformation Gansu Province, Lanzhou, China
| | - Ke Pu
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Xiaojun Liu
- Department of Medical Oncology, The First Hospital of Lanzhou University, Lanzhou, China
| | - Sarah Da Won Bae
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
| | - Romario Nguyen
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
| | - Suyang Bai
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Yi Li
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Liang Qiao
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
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37
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Dai X, Guo X. Decoding and rejuvenating human ageing genomes: Lessons from mosaic chromosomal alterations. Ageing Res Rev 2021; 68:101342. [PMID: 33866012 DOI: 10.1016/j.arr.2021.101342] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2021] [Revised: 04/05/2021] [Accepted: 04/07/2021] [Indexed: 01/10/2023]
Abstract
One of the most curious findings emerged from genome-wide studies over the last decade was that genetic mosaicism is a dominant feature of human ageing genomes. The clonal dominance of genetic mosaicism occurs preceding the physiological and physical ageing and associates with propensity for diseases including cancer, Alzheimer's disease, cardiovascular disease and diabetes. These findings are revolutionizing the ways biologists thinking about health and disease pathogenesis. Among all mosaic mutations in ageing genomes, mosaic chromosomal alterations (mCAs) have the most significant functional consequences because they can produce intercellular genomic variations simultaneously involving dozens to hundreds or even thousands genes, and therefore have most profound effects in human ageing and disease etiology. Here, we provide a comprehensive picture of the landscapes, causes, consequences and rejuvenation of mCAs at multiple scales, from cell to human population, by reviewing data from cytogenetic, genetic and genomic studies in cells, animal models (fly and mouse) and, more frequently, large-cohort populations. A detailed decoding of ageing genomes with a focus on mCAs may yield important insights into the genomic architecture of human ageing, accelerate the risk stratification of age-related diseases (particularly cancers) and development of novel targets and strategies for delaying or rejuvenating human (genome) ageing.
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Affiliation(s)
- Xueqin Dai
- School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, 650500, China
| | - Xihan Guo
- School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, 650500, China; The Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Kunming, Yunnan, 650500, China; Yunnan Environmental Mutagen Society, Kunming, Yunnan, 650500, China.
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38
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Monk R, Connor B. Cell Reprogramming to Model Huntington's Disease: A Comprehensive Review. Cells 2021; 10:cells10071565. [PMID: 34206228 PMCID: PMC8306243 DOI: 10.3390/cells10071565] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2021] [Revised: 06/20/2021] [Accepted: 06/21/2021] [Indexed: 12/16/2022] Open
Abstract
Huntington’s disease (HD) is a neurodegenerative disorder characterized by the progressive decline of motor, cognitive, and psychiatric functions. HD results from an autosomal dominant mutation that causes a trinucleotide CAG repeat expansion and the production of mutant Huntingtin protein (mHTT). This results in the initial selective and progressive loss of medium spiny neurons (MSNs) in the striatum before progressing to involve the whole brain. There are currently no effective treatments to prevent or delay the progression of HD as knowledge into the mechanisms driving the selective degeneration of MSNs has been hindered by a lack of access to live neurons from individuals with HD. The invention of cell reprogramming provides a revolutionary technique for the study, and potential treatment, of neurological conditions. Cell reprogramming technologies allow for the generation of live disease-affected neurons from patients with neurological conditions, becoming a primary technique for modelling these conditions in vitro. The ability to generate HD-affected neurons has widespread applications for investigating the pathogenesis of HD, the identification of new therapeutic targets, and for high-throughput drug screening. Cell reprogramming also offers a potential autologous source of cells for HD cell replacement therapy. This review provides a comprehensive analysis of the use of cell reprogramming to model HD and a discussion on recent advancements in cell reprogramming technologies that will benefit the HD field.
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39
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iPSC Preparation and Epigenetic Memory: Does the Tissue Origin Matter? Cells 2021; 10:cells10061470. [PMID: 34208270 PMCID: PMC8230744 DOI: 10.3390/cells10061470] [Citation(s) in RCA: 53] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 06/06/2021] [Accepted: 06/10/2021] [Indexed: 02/07/2023] Open
Abstract
The production of induced pluripotent stem cells (iPSCs) represent a breakthrough in regenerative medicine, providing new opportunities for understanding basic molecular mechanisms of human development and molecular aspects of degenerative diseases. In contrast to human embryonic stem cells (ESCs), iPSCs do not raise any ethical concerns regarding the onset of human personhood. Still, they present some technical issues related to immune rejection after transplantation and potential tumorigenicity, indicating that more steps forward must be completed to use iPSCs as a viable tool for in vivo tissue regeneration. On the other hand, cell source origin may be pivotal to iPSC generation since residual epigenetic memory could influence the iPSC phenotype and transplantation outcome. In this paper, we first review the impact of reprogramming methods and the choice of the tissue of origin on the epigenetic memory of the iPSCs or their differentiated cells. Next, we describe the importance of induction methods to determine the reprogramming efficiency and avoid integration in the host genome that could alter gene expression. Finally, we compare the significance of the tissue of origin and the inter-individual genetic variation modification that has been lightly evaluated so far, but which significantly impacts reprogramming.
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40
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Anderson NC, Chen PF, Meganathan K, Afshar Saber W, Petersen AJ, Bhattacharyya A, Kroll KL, Sahin M. Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders. Stem Cell Reports 2021; 16:1446-1457. [PMID: 33861989 PMCID: PMC8190574 DOI: 10.1016/j.stemcr.2021.03.025] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 03/18/2021] [Accepted: 03/22/2021] [Indexed: 12/13/2022] Open
Abstract
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors.
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Affiliation(s)
- Nickesha C Anderson
- Department of Neurology, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Pin-Fang Chen
- Department of Neurology, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Kesavan Meganathan
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO 63110, USA
| | - Wardiya Afshar Saber
- Department of Neurology, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | | | - Anita Bhattacharyya
- Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA.
| | - Kristen L Kroll
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO 63110, USA.
| | - Mustafa Sahin
- Department of Neurology, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
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Ray A, Joshi JM, Sundaravadivelu PK, Raina K, Lenka N, Kaveeshwar V, Thummer RP. An Overview on Promising Somatic Cell Sources Utilized for the Efficient Generation of Induced Pluripotent Stem Cells. Stem Cell Rev Rep 2021; 17:1954-1974. [PMID: 34100193 DOI: 10.1007/s12015-021-10200-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/02/2021] [Indexed: 01/19/2023]
Abstract
Human induced Pluripotent Stem Cells (iPSCs) have enormous potential in understanding developmental biology, disease modeling, drug discovery, and regenerative medicine. The initial human iPSC studies used fibroblasts as a starting cell source to reprogram them; however, it has been identified to be a less appealing somatic cell source by numerous studies due to various reasons. One of the important criteria to achieve efficient reprogramming is determining an appropriate starting somatic cell type to induce pluripotency since the cellular source has a major influence on the reprogramming efficiency, kinetics, and quality of iPSCs. Therefore, numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. This review provides an overview of promising easily accessible somatic cell sources isolated in non-invasive or minimally invasive manner such as keratinocytes, urine cells, and peripheral blood mononuclear cells used for the generation of human iPSCs derived from healthy and diseased subjects. Notably, iPSCs generated from one of these cell types derived from the patient will offer ethical and clinical advantages. In addition, these promising somatic cell sources have the potential to efficiently generate bona fide iPSCs with improved reprogramming efficiency and faster kinetics. This knowledge will help in establishing strategies for safe and efficient reprogramming and the generation of patient-specific iPSCs from these cell types.
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Affiliation(s)
- Arnab Ray
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Jahnavy Madhukar Joshi
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, 580009, Karnataka, India
| | - Pradeep Kumar Sundaravadivelu
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Khyati Raina
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Nibedita Lenka
- National Centre for Cell Science, S. P. Pune University Campus, Pune - 411007, Ganeshkhind, Maharashtra, India
| | - Vishwas Kaveeshwar
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, 580009, Karnataka, India.
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
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Güney-Esken G, Erol ÖD, Pervin B, Gürhan Sevinç G, Önder T, Bilgiç E, Korkusuz P, Günel-Özcan A, Uçkan-Çetinkaya D, Aerts-Kaya F. Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells. Stem Cell Res Ther 2021; 12:287. [PMID: 33985578 PMCID: PMC8117610 DOI: 10.1186/s13287-021-02364-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Accepted: 04/29/2021] [Indexed: 11/17/2022] Open
Abstract
BACKGROUND Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2. METHODS Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media. RESULTS All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage. CONCLUSIONS Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.
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Affiliation(s)
- Gülen Güney-Esken
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Özgür Doğuş Erol
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Burcu Pervin
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Gülben Gürhan Sevinç
- School of Medicine, Research Center for Translational Medicine, Koç University, Istanbul, Turkey
| | - Tamer Önder
- School of Medicine, Research Center for Translational Medicine, Koç University, Istanbul, Turkey
| | - Elif Bilgiç
- Faculty of Medicine, Department of Histology and Embryology, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Petek Korkusuz
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Faculty of Medicine, Department of Histology and Embryology, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Ayşen Günel-Özcan
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
| | - Duygu Uçkan-Çetinkaya
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey
- Faculty of Medicine, Department of Pediatrics, Division of Hematology, Hacettepe University, Ankara, Turkey
| | - Fatima Aerts-Kaya
- Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey.
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey.
- Laboratory Animals Research and Application Center (HUDHAM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey.
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Wang Y, Bae T, Thorpe J, Sherman MA, Jones AG, Cho S, Daily K, Dou Y, Ganz J, Galor A, Lobon I, Pattni R, Rosenbluh C, Tomasi S, Tomasini L, Yang X, Zhou B, Akbarian S, Ball LL, Bizzotto S, Emery SB, Doan R, Fasching L, Jang Y, Juan D, Lizano E, Luquette LJ, Moldovan JB, Narurkar R, Oetjens MT, Rodin RE, Sekar S, Shin JH, Soriano E, Straub RE, Zhou W, Chess A, Gleeson JG, Marquès-Bonet T, Park PJ, Peters MA, Pevsner J, Walsh CA, Weinberger DR, Vaccarino FM, Moran JV, Urban AE, Kidd JM, Mills RE, Abyzov A. Comprehensive identification of somatic nucleotide variants in human brain tissue. Genome Biol 2021; 22:92. [PMID: 33781308 PMCID: PMC8006362 DOI: 10.1186/s13059-021-02285-3] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 02/01/2021] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.
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Affiliation(s)
- Yifan Wang
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, 100 Washtenaw Avenue, Ann Arbor, MI, 48109, USA
| | - Taejeong Bae
- Department of Health Sciences Research, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA
| | - Jeremy Thorpe
- Program in Biochemistry, Cellular and Molecular Biology, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA
| | - Maxwell A Sherman
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
- MIT Department of Electrical Engineering and Computer Science, Cambridge, MA, USA
| | - Attila G Jones
- Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Sean Cho
- Department of Neurology, Kennedy Krieger Institute, Baltimore, MD, 21205, USA
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA
- Present Address: Arcus Biosciences, Hayward, CA, 94545, USA
| | | | - Yanmei Dou
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
| | - Javier Ganz
- Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, 02115, USA
- Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA, 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA
| | - Alon Galor
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
| | - Irene Lobon
- Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), PRBB, 08003, Barcelona, Catalonia, Spain
- Department of Cell Biology, Physiology and Immunology, and Institute of Neurosciences, University of Barcelona, 08028, Barcelona, Spain
| | - Reenal Pattni
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, 94305, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Chaggai Rosenbluh
- Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Simone Tomasi
- Child Study Center, Yale University, New Haven, CT, 06520, USA
| | - Livia Tomasini
- Child Study Center, Yale University, New Haven, CT, 06520, USA
| | - Xiaoxu Yang
- Department of Neurosciences, University of California San Diego, La Jolla, CA, USA
- Rady Children's Institute for Genomic Medicine, San Diego, CA, USA
| | - Bo Zhou
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, 94305, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Schahram Akbarian
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Laurel L Ball
- Department of Neurosciences, University of California San Diego, La Jolla, CA, USA
- Rady Children's Institute for Genomic Medicine, San Diego, CA, USA
| | - Sara Bizzotto
- Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, 02115, USA
- Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA, 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA
| | - Sarah B Emery
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - Ryan Doan
- Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, 02115, USA
- Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA, 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA
| | - Liana Fasching
- Child Study Center, Yale University, New Haven, CT, 06520, USA
| | - Yeongjun Jang
- Department of Health Sciences Research, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA
| | - David Juan
- Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), PRBB, 08003, Barcelona, Catalonia, Spain
| | - Esther Lizano
- Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), PRBB, 08003, Barcelona, Catalonia, Spain
| | - Lovelace J Luquette
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
| | - John B Moldovan
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - Rujuta Narurkar
- Lieber Institute for Brain Development, Baltimore, MD, 21205, USA
| | - Matthew T Oetjens
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - Rachel E Rodin
- Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, 02115, USA
- Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA, 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA
| | - Shobana Sekar
- Department of Health Sciences Research, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA
| | - Joo Heon Shin
- Lieber Institute for Brain Development, Baltimore, MD, 21205, USA
- Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Eduardo Soriano
- Department of Cell Biology, Physiology and Immunology, and Institute of Neurosciences, University of Barcelona, 08028, Barcelona, Spain
- Vall d'Hebron Institut de Recerca, 08035, Barcelona, Spain
- Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031, Madrid, Spain
- ICREA Academia, 08010 Barcelona, Spain
| | - Richard E Straub
- Lieber Institute for Brain Development, Baltimore, MD, 21205, USA
| | - Weichen Zhou
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, 100 Washtenaw Avenue, Ann Arbor, MI, 48109, USA
| | - Andrew Chess
- Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Icahn Institute for Data Science and Genomic Technologies, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Joseph G Gleeson
- Department of Neurosciences, University of California San Diego, La Jolla, CA, USA
- Rady Children's Institute for Genomic Medicine, San Diego, CA, USA
| | - Tomas Marquès-Bonet
- Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), PRBB, 08003, Barcelona, Catalonia, Spain
- Catalan Institution of Research and Advanced Studies (ICREA), 08010, Barcelona, Spain
- CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), 08036, Barcelona, Spain
- Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, Barcelona, Spain
| | - Peter J Park
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
| | | | - Jonathan Pevsner
- Department of Neurology, Kennedy Krieger Institute, Baltimore, MD, 21205, USA
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA
| | - Christopher A Walsh
- Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, 02115, USA
- Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA, 02115, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA
| | - Daniel R Weinberger
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA
- Lieber Institute for Brain Development, Baltimore, MD, 21205, USA
- Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA
- Department of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA
- Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Flora M Vaccarino
- Child Study Center, Yale University, New Haven, CT, 06520, USA
- Department of Neuroscience, Yale University, New Haven, 06520, CT, USA
| | - John V Moran
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - Alexander E Urban
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, 94305, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA
- Tashia and John Morgridge Faculty Scholar, Stanford Child Health Research Institute, Stanford, CA, 94305, USA
| | - Jeffrey M Kidd
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, 100 Washtenaw Avenue, Ann Arbor, MI, 48109, USA
| | - Ryan E Mills
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, 100 Washtenaw Avenue, Ann Arbor, MI, 48109, USA
| | - Alexej Abyzov
- Department of Health Sciences Research, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA.
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Jourdon A, Vaccarino FM. One for All: A Pooled Approach to Classify Functional Impacts of Multiple Mutations. Cell Stem Cell 2021; 27:1-3. [PMID: 32619508 DOI: 10.1016/j.stem.2020.06.016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Understanding common biological consequences of heterogenous mutations in complex polygenic conditions is challenging. In this issue of Cell Stem Cell, Cederquist et al. (2020) implement an in vitro pooled assay where 30 high-confidence ASD mutations engineered in subclones of a human pluripotent stem cell line can be investigated in parallel to reveal their effects on prefrontal cortex neurogenesis.
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Affiliation(s)
| | - Flora M Vaccarino
- Child Study Center, Yale University, New Haven, CT 06520, USA; Department of Neuroscience, Yale University, New Haven, CT 06520, USA.
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45
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Stertz L, Di Re J, Pei G, Fries GR, Mendez E, Li S, Smith-Callahan L, Raventos H, Tipo J, Cherukuru R, Zhao Z, Liu Y, Jia P, Laezza F, Walss-Bass C. Convergent genomic and pharmacological evidence of PI3K/GSK3 signaling alterations in neurons from schizophrenia patients. Neuropsychopharmacology 2021; 46:673-682. [PMID: 33288841 PMCID: PMC8027596 DOI: 10.1038/s41386-020-00924-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 10/30/2020] [Accepted: 11/02/2020] [Indexed: 12/12/2022]
Abstract
Human-induced pluripotent stem cells (hiPSCs) allow for the establishment of brain cellular models of psychiatric disorders that account for a patient's genetic background. Here, we conducted an RNA-sequencing profiling study of hiPSC-derived cell lines from schizophrenia (SCZ) subjects, most of which are from a multiplex family, from the population isolate of the Central Valley of Costa Rica. hiPSCs, neural precursor cells, and cortical neurons derived from six healthy controls and seven SCZ subjects were generated using standard methodology. Transcriptome from these cells was obtained using Illumina HiSeq 2500, and differential expression analyses were performed using DESeq2 (|fold change|>1.5 and false discovery rate < 0.3), in patients compared to controls. We identified 454 differentially expressed genes in hiPSC-derived neurons, enriched in pathways including phosphoinositide 3-kinase/glycogen synthase kinase 3 (PI3K/GSK3) signaling, with serum-glucocorticoid kinase 1 (SGK1), an inhibitor of glycogen synthase kinase 3β, as part of this pathway. We further found that pharmacological inhibition of downstream effectors of the PI3K/GSK3 pathway, SGK1 and GSK3, induced alterations in levels of neurite markers βIII tubulin and fibroblast growth factor 12, with differential effects in patients compared to controls. While demonstrating the utility of hiPSCs derived from multiplex families to identify significant cell-specific gene network alterations in SCZ, these studies support a role for disruption of PI3K/GSK3 signaling as a risk factor for SCZ.
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Affiliation(s)
- Laura Stertz
- Louis A. Faillace, MD, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Jessica Di Re
- Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA
| | - Guangsheng Pei
- Center for Precision Health, School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Gabriel R Fries
- Louis A. Faillace, MD, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
- Center for Precision Health, School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Emily Mendez
- Louis A. Faillace, MD, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Shenglan Li
- Institute of Molecular Medicine for the Prevention of Human Diseases, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Laura Smith-Callahan
- Institute of Molecular Medicine for the Prevention of Human Diseases, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Henriette Raventos
- Centro de Investigacion en Biologia Celular y Molecular, Universidad de Costa Rica, San Jose, Costa Rica
| | - Jerricho Tipo
- School of Medicine, University of Texas Medical Branch, Galveston, TX, USA
| | - Rohan Cherukuru
- Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA
| | - Zhongming Zhao
- Louis A. Faillace, MD, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
- Center for Precision Health, School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Ying Liu
- Institute of Molecular Medicine for the Prevention of Human Diseases, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Peilin Jia
- Center for Precision Health, School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Fernanda Laezza
- Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA
| | - Consuelo Walss-Bass
- Louis A. Faillace, MD, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA.
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UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin. PLoS Genet 2021; 17:e1009302. [PMID: 33444353 PMCID: PMC7808690 DOI: 10.1371/journal.pgen.1009302] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Accepted: 12/07/2020] [Indexed: 02/06/2023] Open
Abstract
Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis. Skin forms the first barrier against a variety of environmental toxins and DNA damaging agents. Additionally, DNA of skin cells suffer from endogenous damage and errors during replication. Altogether, these lesions cause a variety of genome changes resulting in disease including cancer. However, the accurate measurement of the range and complete spectrum of genome changes in healthy skin was missing due to technical or biological limitations of prior studies. We present here accurate measurements of the various types of somatic genome changes that we found in skin fibroblasts and melanocytes from 21 donors ranging in ages from 25 to 79 years, which allowed to distinguish age related from age independent changes. Our cohort contains both White and African American donors, allowing an estimation of the impacts of skin color on mutagenesis. As a result, we revealed the complete spectrum and determined the range of somatic genome changes and their etiologies in healthy human skin fibroblasts and melanocytes and highlighted molecular mechanisms underlying these changes. Therefore, our study introduces a base line for defining disease levels of genome instability in skin.
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47
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Strategies for Cancer Immunotherapy Using Induced Pluripotency Stem Cells-Based Vaccines. Cancers (Basel) 2020; 12:cancers12123581. [PMID: 33266109 PMCID: PMC7760556 DOI: 10.3390/cancers12123581] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2020] [Revised: 11/24/2020] [Accepted: 11/27/2020] [Indexed: 12/14/2022] Open
Abstract
Despite improvements in cancer therapy, metastatic solid tumors remain largely incurable. Immunotherapy has emerged as a pioneering and promising approach for cancer therapy and management, and in particular intended for advanced tumors unresponsive to current therapeutics. In cancer immunotherapy, components of the immune system are exploited to eliminate cancer cells and treat patients. The recent clinical successes of immune checkpoint blockade and chimeric antigen receptor T cell therapies represent a turning point in cancer treatment. Despite their potential success, current approaches depend on efficient tumor antigen presentation which are often inaccessible, and most tumors turn refractory to current immunotherapy. Patient-derived induced pluripotent stem cells (iPSCs) have been shown to share several characteristics with cancer (stem) cells (CSCs), eliciting a specific anti-tumoral response when injected in rodent cancer models. Indeed, artificial cellular reprogramming has been widely compared to the biogenesis of CSCs. Here, we will discuss the state-of-the-art on the potential implication of cellular reprogramming and iPSCs for the design of patient-specific immunotherapeutic strategies, debating the similarities between iPSCs and cancer cells and introducing potential strategies that could enhance the efficiency and therapeutic potential of iPSCs-based cancer vaccines.
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48
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Zou Z, Long X, Zhao Q, Zheng Y, Song M, Ma S, Jing Y, Wang S, He Y, Esteban CR, Yu N, Huang J, Chan P, Chen T, Izpisua Belmonte JC, Zhang W, Qu J, Liu GH. A Single-Cell Transcriptomic Atlas of Human Skin Aging. Dev Cell 2020; 56:383-397.e8. [PMID: 33238152 DOI: 10.1016/j.devcel.2020.11.002] [Citation(s) in RCA: 194] [Impact Index Per Article: 38.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 09/26/2020] [Accepted: 11/02/2020] [Indexed: 12/22/2022]
Abstract
Skin undergoes constant self-renewal, and its functional decline is a visible consequence of aging. Understanding human skin aging requires in-depth knowledge of the molecular and functional properties of various skin cell types. We performed single-cell RNA sequencing of human eyelid skin from healthy individuals across different ages and identified eleven canonical cell types, as well as six subpopulations of basal cells. Further analysis revealed progressive accumulation of photoaging-related changes and increased chronic inflammation with age. Transcriptional factors involved in the developmental process underwent early-onset decline during aging. Furthermore, inhibition of key transcription factors HES1 in fibroblasts and KLF6 in keratinocytes not only compromised cell proliferation, but also increased inflammation and cellular senescence during aging. Lastly, we found that genetic activation of HES1 or pharmacological treatment with quercetin alleviated cellular senescence of dermal fibroblasts. These findings provide a single-cell molecular framework of human skin aging, providing a rich resource for developing therapeutic strategies against aging-related skin disorders.
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Affiliation(s)
- Zhiran Zou
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiao Long
- Division of Plastic Surgery, Peking Union Medical College Hospital, Beijing 100032, China
| | - Qian Zhao
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing 100053, China
| | - Yandong Zheng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Moshi Song
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuai Ma
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yaobin Jing
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Si Wang
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing 100053, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yifang He
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | | | - Nanze Yu
- Division of Plastic Surgery, Peking Union Medical College Hospital, Beijing 100032, China
| | - Jiuzuo Huang
- Division of Plastic Surgery, Peking Union Medical College Hospital, Beijing 100032, China
| | - Piu Chan
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing 100053, China
| | - Ting Chen
- National Institute of Biological Sciences, Beijing 102206, China
| | | | - Weiqi Zhang
- CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; China National Center for Bioinformation, Beijing 100101, China.
| | - Jing Qu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Guang-Hui Liu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing 100053, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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de Leeuw VC, van Oostrom CTM, Imholz S, Piersma AH, Hessel EVS, Dollé MET. Going Back and Forth: Episomal Vector Reprogramming of Peripheral Blood Mononuclear Cells to Induced Pluripotent Stem Cells and Subsequent Differentiation into Cardiomyocytes and Neuron-Astrocyte Co-cultures. Cell Reprogram 2020; 22:300-310. [PMID: 33146557 PMCID: PMC7757589 DOI: 10.1089/cell.2020.0040] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Human induced pluripotent stem cells (iPSCs) can capture the diversity in the general human population as well as provide deeper insight in cellular mechanisms. This makes them suitable to study both fundamental and applied research subjects, such as disease modeling, gene-environment interactions, personalized medicine, and chemical toxicity. In an independent laboratory, we were able to generate iPSCs originating from human peripheral blood mononuclear cells according to a modified version of a temporal episomal vector (EV)-based induction method. The iPSCs could subsequently be differentiated into two different lineages: mesoderm-derived cardiomyocytes and ectoderm-derived neuron-astrocyte co-cultures. It was shown that the neuron-astrocyte culture developed a mature phenotype within the course of five weeks and depending on the medium composition, network formation and neuron-astrocyte cell ratios could be modified. Although previously it has been described that iPSCs generated with this EV-based induction protocol could differentiate to mesenchymal stem cells, hepatocytes, cardiomyocytes, and basic neuronal cultures, we now demonstrate differentiation into a culture containing both neurons and astrocytes.
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Affiliation(s)
- Victoria C de Leeuw
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.,Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands
| | - Conny T M van Oostrom
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
| | - Sandra Imholz
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
| | - Aldert H Piersma
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.,Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands
| | - Ellen V S Hessel
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
| | - Martijn E T Dollé
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
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Chromosomal aberration arises during somatic reprogramming to pluripotent stem cells. Cell Div 2020; 15:12. [PMID: 33292330 PMCID: PMC7641821 DOI: 10.1186/s13008-020-00068-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Accepted: 10/26/2020] [Indexed: 12/18/2022] Open
Abstract
Background Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) has opened new therapeutic possibilities. However, karyotypic abnormalities detected in iPSCs compromised their utility, especially chromosomal aberrations found at early passages raised serious safety concerns. The mechanism underlying the chromosomal abnormality in early-passage iPSCs is not known. Methods Human dermal fibroblasts (HDFs) were stimulated with KMOS (KLF4, cMYC, OCT4 and SOX2) proteins to enhance their proliferative capacity and many vigorous clones were obtained. Clonal reprogramming was carried out by KMOS mRNAs transfection to confirm the ‘chromosomal mutagenicity’ of reprogramming process. Subculturing was performed to examine karyotypic stability of iPSCs after the re-establishment of stemness. And antioxidant N-acetyl-cysteine (NAC) was added to the culture medium for further confirmming the mutagenicity in the first few days of reprogramming. Results Chromosomal aberrations were found in a small percentage of newly induced iPS clones by reprogramming transcription factors. Clonal reprogramming ruled out the aberrant chromosomes inherited from rare karyotypically abnormal parental cell subpopulation. More importantly, the antioxidant NAC effectively reduced the occurrence of chromosomal aberrations at the early stage of reprogramming. Once iPS cell lines were established, they restored karyotypic stability in subsequent subculturing. Conclusions Our results provided the first line of evidence for the ‘chromosomal mutagenicity’ of reprogramming process.
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