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Francis A, McKibben LA, Dwivedi Y. Early-Life Adversity-Induced Epigenetic Reprogramming of Prefrontal Cortex in Rats Subjected to Maternal Separation. BIOLOGICAL PSYCHIATRY GLOBAL OPEN SCIENCE 2025; 5:100487. [PMID: 40342557 PMCID: PMC12060456 DOI: 10.1016/j.bpsgos.2025.100487] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Revised: 02/21/2025] [Accepted: 03/01/2025] [Indexed: 05/11/2025] Open
Abstract
Background Early-life adversity (ELA) can lead to long-lasting behavioral and neurobiological changes through epigenetic mechanisms. In this study, we comprehensively mapped genome-wide DNA methylation in the prefrontal cortex of rats following maternal separation (MS). Methods Rat pups were separated from their mother for 180 minutes/day from postnatal days (PNDs) 1 to 14 and tested for depressive- and anxiety-like behavior during adulthood (PNDs 80-89). Genome-wide DNA methylation, corresponding functional analyses, and transcription factor binding sites (TFBSs) were performed using reduced-representation bisulfite sequencing, focusing on differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and non-CpG sites. Results Both male and female MS rats showed a significant decrease in sucrose preference. Principal component and multidimensional scaling analyses did not show differences in the methylation data between male and female rats, prompting us to combine them in subsequent analyses. A total of 33,905 DMCs and 151 DMRs were identified in the MS group. The functional analysis of the dysregulated genes by DMCs and DMRs in the promoter or gene body revealed gene enrichment involved in neurodevelopment, synaptic plasticity, and stress response. Key genes with altered methylation included Dnmt3a/b, Notch1, Mapk14, and calcium channel subunits. Gene network analysis revealed interactions among ribosomal, MAPK (mitogen-activated protein kinase), and glutamatergic pathway genes. An enrichment of Elk1 TFBSs was particularly noted within the DMR. Additionally, differential non-CpG methylation, specifically at CHH (H = C/T/A) sites, dysregulated the Wnt pathway genes. Conclusions Our findings expand our understanding of the molecular mechanisms that underlie the long-term effects of ELA and identify potential biomarkers for stress-related psychiatric disorders.
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Affiliation(s)
- Aleena Francis
- Department of Psychiatry and Behavioral Neurobiology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
| | - Lauren Allen McKibben
- Institute for Trauma Recovery, Department of Anesthesiology, University of North Carolina School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Yogesh Dwivedi
- Department of Psychiatry and Behavioral Neurobiology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
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2
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Zhang YX, Zhou Y, Xiong YY, Li YM. Beyond skin deep: Revealing the essence of iPS cell-generated skin organoids in regeneration. Burns 2024; 50:107194. [PMID: 39317530 DOI: 10.1016/j.burns.2024.06.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2023] [Revised: 03/13/2024] [Accepted: 06/23/2024] [Indexed: 09/26/2024]
Abstract
Various methods have been used for in vivo and in vitro skin regeneration, including stem cell therapy, tissue engineering, 3D printing, and platelet-rich plasma (PRP) injection therapy. However, these approaches are rooted in the existing knowledge of skin structures, which overlook the normal physiological processes of skin development and fall short of replicating the skin's regenerative processes outside the body. This comprehensive review primarily focuses on skin organoids derived from human pluripotent stem cells, which have the capacity to regenerate human skin tissue by restoring the embryonic skin structure, thus offering a novel avenue for producing in vitro skin substitutes. Furthermore, they contribute to the repair of damaged skin lesions in patients with systemic sclerosis or severe burns. Particular emphasis will be placed on the origins, generations, and applications of skin organoids, especially in dermatology, and the challenges that must be addressed before clinical implementation.
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Affiliation(s)
- Yu-Xuan Zhang
- Institute of Regenerative Medicine, and Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China
| | - Yuan Zhou
- Institute of Regenerative Medicine, and Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China
| | - Yu-Yun Xiong
- Institute of Regenerative Medicine, and Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China.
| | - Yu-Mei Li
- Institute of Regenerative Medicine, and Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China.
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Scatturice LA, Vázquez N, Strobl-Mazzulla PH. miR-137 confers robustness to the territorial restriction of the neural plate border. Development 2024; 151:dev202344. [PMID: 38828854 DOI: 10.1242/dev.202344] [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: 09/08/2023] [Accepted: 05/20/2024] [Indexed: 06/05/2024]
Abstract
The neural plate border (NPB) of vertebrate embryos is segregated from the neural plate (NP) and epidermal regions, and comprises an intermingled group of progenitors with multiple fate potential. Recent studies have shown that, during the gastrula stage, TFAP2A acts as a pioneer factor in remodeling the epigenetic landscape required to activate components of the NPB induction program. Here, we show that chick Tfap2a has two highly conserved binding sites for miR-137, and both display a reciprocal expression pattern at the NPB and NP, respectively. In addition, ectopic miR-137 expression reduced TFAP2A, whereas its functional inhibition expanded their territorial distribution overlapping with PAX7. Furthermore, we demonstrate that loss of the de novo DNA methyltransferase DNMT3A expanded miR-137 expression to the NPB. Bisulfite sequencing revealed a markedly elevated presence of non-canonical CpH methylation within the miR-137 promoter region when comparing NPB and NP samples. Our findings show that miR-137 contributes to the robustness of NPB territorial restriction in vertebrate development.
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Affiliation(s)
- Luciana A Scatturice
- Laboratory of Developmental Biology, Instituto Tecnológico de Chascomús (CONICET-UNSAM). Escuela de Bio y Nanotecnologías (UNSAM), Chascomús, Buenos Aires 7130, Argentina
| | - Nicolás Vázquez
- Laboratory of Developmental Biology, Instituto Tecnológico de Chascomús (CONICET-UNSAM). Escuela de Bio y Nanotecnologías (UNSAM), Chascomús, Buenos Aires 7130, Argentina
| | - Pablo H Strobl-Mazzulla
- Laboratory of Developmental Biology, Instituto Tecnológico de Chascomús (CONICET-UNSAM). Escuela de Bio y Nanotecnologías (UNSAM), Chascomús, Buenos Aires 7130, Argentina
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Araki R, Suga T, Hoki Y, Imadome K, Sunayama M, Kamimura S, Fujita M, Abe M. iPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms. Nat Commun 2024; 15:4946. [PMID: 38862540 PMCID: PMC11166658 DOI: 10.1038/s41467-024-49335-5] [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: 04/17/2023] [Accepted: 05/31/2024] [Indexed: 06/13/2024] Open
Abstract
Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.
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Affiliation(s)
- Ryoko Araki
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan.
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan.
| | - Tomo Suga
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Yuko Hoki
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Kaori Imadome
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Misato Sunayama
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Satoshi Kamimura
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Mayumi Fujita
- Stem Cell Biology Team, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Chiba, Japan
- Department of Radiation Regulatory Science Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - Masumi Abe
- Institute for Quantum Medical Science, National Institutes for Quantum Science and Technology, Chiba, Japan.
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Cerneckis J, Cai H, Shi Y. Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications. Signal Transduct Target Ther 2024; 9:112. [PMID: 38670977 PMCID: PMC11053163 DOI: 10.1038/s41392-024-01809-0] [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: 07/28/2023] [Revised: 03/09/2024] [Accepted: 03/17/2024] [Indexed: 04/28/2024] Open
Abstract
The induced pluripotent stem cell (iPSC) technology has transformed in vitro research and holds great promise to advance regenerative medicine. iPSCs have the capacity for an almost unlimited expansion, are amenable to genetic engineering, and can be differentiated into most somatic cell types. iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies. In this review, we outline key developments in the iPSC field and highlight the immense versatility of the iPSC technology for in vitro modeling and therapeutic applications. We begin by discussing the pivotal discoveries that revealed the potential of a somatic cell nucleus for reprogramming and led to successful generation of iPSCs. We consider the molecular mechanisms and dynamics of somatic cell reprogramming as well as the numerous methods available to induce pluripotency. Subsequently, we discuss various iPSC-based cellular models, from mono-cultures of a single cell type to complex three-dimensional organoids, and how these models can be applied to elucidate the mechanisms of human development and diseases. We use examples of neurological disorders, coronavirus disease 2019 (COVID-19), and cancer to highlight the diversity of disease-specific phenotypes that can be modeled using iPSC-derived cells. We also consider how iPSC-derived cellular models can be used in high-throughput drug screening and drug toxicity studies. Finally, we discuss the process of developing autologous and allogeneic iPSC-based cell therapies and their potential to alleviate human diseases.
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Affiliation(s)
- Jonas Cerneckis
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Hongxia Cai
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Yanhong Shi
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
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Umeyama T, Matsuda T, Nakashima K. Lineage Reprogramming: Genetic, Chemical, and Physical Cues for Cell Fate Conversion with a Focus on Neuronal Direct Reprogramming and Pluripotency Reprogramming. Cells 2024; 13:707. [PMID: 38667322 PMCID: PMC11049106 DOI: 10.3390/cells13080707] [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: 04/02/2024] [Revised: 04/16/2024] [Accepted: 04/17/2024] [Indexed: 04/28/2024] Open
Abstract
Although lineage reprogramming from one cell type to another is becoming a breakthrough technology for cell-based therapy, several limitations remain to be overcome, including the low conversion efficiency and subtype specificity. To address these, many studies have been conducted using genetics, chemistry, physics, and cell biology to control transcriptional networks, signaling cascades, and epigenetic modifications during reprogramming. Here, we summarize recent advances in cellular reprogramming and discuss future directions.
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Affiliation(s)
- Taichi Umeyama
- Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 819-0395, Japan
| | - Taito Matsuda
- Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 819-0395, Japan
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7
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Edwards MM, Wang N, Massey DJ, Bhatele S, Egli D, Koren A. Incomplete reprogramming of DNA replication timing in induced pluripotent stem cells. Cell Rep 2024; 43:113664. [PMID: 38194345 PMCID: PMC11231959 DOI: 10.1016/j.celrep.2023.113664] [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: 06/22/2023] [Revised: 10/27/2023] [Accepted: 12/21/2023] [Indexed: 01/10/2024] Open
Abstract
Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.
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Affiliation(s)
- Matthew M Edwards
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
| | - Ning Wang
- Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA; Columbia University Stem Cell Initiative, New York, NY 10032, USA
| | - Dashiell J Massey
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
| | - Sakshi Bhatele
- Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA; Columbia University Stem Cell Initiative, New York, NY 10032, USA
| | - Dieter Egli
- Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA; Columbia University Stem Cell Initiative, New York, NY 10032, USA.
| | - Amnon Koren
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA; Department of Molecular and Cellular Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA.
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Camões dos Santos J, Appleton C, Cazaux Mateus F, Covas R, Bekman EP, da Rocha ST. Stem cell models of Angelman syndrome. Front Cell Dev Biol 2023; 11:1274040. [PMID: 37928900 PMCID: PMC10620611 DOI: 10.3389/fcell.2023.1274040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 10/02/2023] [Indexed: 11/07/2023] Open
Abstract
Angelman syndrome (AS) is an imprinted neurodevelopmental disorder that lacks a cure, characterized by developmental delay, intellectual impairment, seizures, ataxia, and paroxysmal laughter. The condition arises due to the loss of the maternally inherited copy of the UBE3A gene in neurons. The paternally inherited UBE3A allele is unable to compensate because it is silenced by the expression of an antisense transcript (UBE3A-ATS) on the paternal chromosome. UBE3A, encoding enigmatic E3 ubiquitin ligase variants, regulates target proteins by either modifying their properties/functions or leading them to degradation through the proteasome. Over time, animal models, particularly the Ube3a mat-/pat+ Knock-Out (KO) mice, have significantly contributed to our understanding of the molecular mechanisms underlying AS. However, a shift toward human pluripotent stem cell models (PSCs), such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has gained momentum. These stem cell models accurately capture human genetic and cellular characteristics, offering an alternative or a complement to animal experimentation. Human stem cells possess the remarkable ability to recapitulate neurogenesis and generate "brain-in-a-dish" models, making them valuable tools for studying neurodevelopmental disorders like AS. In this review, we provide an overview of the current state-of-the-art human stem cell models of AS and explore their potential to become the preclinical models of choice for drug screening and development, thus propelling AS therapeutic advancements and improving the lives of affected individuals.
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Affiliation(s)
- João Camões dos Santos
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Carolina Appleton
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Department of Animal Biology, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal
| | - Francisca Cazaux Mateus
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Rita Covas
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Evguenia Pavlovna Bekman
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- The Egas Moniz Center for Interdisciplinary Research (CiiEM), Caparica, Portugal
| | - Simão Teixeira da Rocha
- iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
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9
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Moon H, Kim B, Kwon I, Oh Y. Challenges involved in cell therapy for Parkinson's disease using human pluripotent stem cells. Front Cell Dev Biol 2023; 11:1288168. [PMID: 37886394 PMCID: PMC10598731 DOI: 10.3389/fcell.2023.1288168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Accepted: 09/25/2023] [Indexed: 10/28/2023] Open
Abstract
Neurons derived from human pluripotent stem cells (hPSCs) provide a valuable tool for studying human neural development and neurodegenerative diseases. The investigation of hPSC-based cell therapy, involving the differentiation of hPSCs into target cells and their transplantation into affected regions, is of particular interest. One neurodegenerative disease that is being extensively studied for hPSC-based cell therapy is Parkinson's disease (PD), the second most common among humans. Various research groups are focused on differentiating hPSCs into ventral midbrain dopaminergic (vmDA) progenitors, which have the potential to further differentiate into neurons closely resembling DA neurons found in the substantia nigra pars compacta (SNpc) after transplantation, providing a promising treatment option for PD. In vivo experiments, where hPSC-derived vmDA progenitor cells were transplanted into the striatum or SNpc of animal PD models, the transplanted cells demonstrated stable engraftment and resulted in behavioral recovery in the transplanted animals. Several differentiation protocols have been developed for this specific cell therapy. However, the lack of a reliable live-cell lineage identification method presents a significant obstacle in confirming the precise lineage of the differentiated cells intended for transplantation, as well as identifying potential contamination by non-vmDA progenitors. This deficiency increases the risk of adverse effects such as dyskinesias and tumorigenicity, highlighting the importance of addressing this issue before proceeding with transplantation. Ensuring the differentiation of hPSCs into the target cell lineage is a crucial step to guarantee precise therapeutic effects in cell therapy. To underscore the significance of lineage identification, this review focuses on the differentiation protocols of hPSC-derived vmDA progenitors developed by various research groups for PD treatment. Moreover, in vivo experimental results following transplantation were carefully analyzed. The encouraging outcomes from these experiments demonstrate the potential efficacy and safety of hPSC-derived vmDA progenitors for PD cell therapy. Additionally, the results of clinical trials involving the use of hPSC-derived vmDA progenitors for PD treatment were briefly reviewed, shedding light on the progress and challenges faced in translating this promising therapy into clinical practice.
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Affiliation(s)
- Heechang Moon
- Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
| | - Bokwang Kim
- Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
| | - Inbeom Kwon
- Department of Medicine, College of Medicine, Hanyang University, Seoul, Republic of Korea
| | - Yohan Oh
- Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
- Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Republic of Korea
- Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, Republic of Korea
- Hanyang Institute of Advanced BioConvergence, Hanyang University, Seoul, Republic of Korea
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10
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A way to wipe a cell's memory. Nature 2023:10.1038/d41586-023-02381-3. [PMID: 37773282 DOI: 10.1038/d41586-023-02381-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/01/2023]
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11
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Buckberry S, Liu X, Poppe D, Tan JP, Sun G, Chen J, Nguyen TV, de Mendoza A, Pflueger J, Frazer T, Vargas-Landín DB, Paynter JM, Smits N, Liu N, Ouyang JF, Rossello FJ, Chy HS, Rackham OJL, Laslett AL, Breen J, Faulkner GJ, Nefzger CM, Polo JM, Lister R. Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature 2023; 620:863-872. [PMID: 37587336 PMCID: PMC10447250 DOI: 10.1038/s41586-023-06424-7] [Citation(s) in RCA: 35] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 07/11/2023] [Indexed: 08/18/2023]
Abstract
Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
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Affiliation(s)
- Sam Buckberry
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
- Telethon Kids Institute, Perth, Western Australia, Australia
- John Curtin School of Medical Research, College of Health and Medicine, Australian National University, Canberra, Australian Capital Territory, Australia
| | - Xiaodong Liu
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
- School of Life Sciences, Westlake University, Hangzhou, China
- Research Center for Industries of the Future, Westlake University, Hangzhou, China
- Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Westlake Institute for Advanced Study, Hangzhou, China
| | - Daniel Poppe
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
| | - Jia Ping Tan
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
| | - Guizhi Sun
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
| | - Joseph Chen
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
| | - Trung Viet Nguyen
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
| | - Alex de Mendoza
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
- School of Biological and Behavioural Sciences, Queen Mary University of London, London, UK
| | - Jahnvi Pflueger
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
| | - Thomas Frazer
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
| | - Dulce B Vargas-Landín
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
| | - Jacob M Paynter
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
| | - Nathan Smits
- Mater Research Institute, University of Queensland, Brisbane, Queensland, Australia
| | - Ning Liu
- South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia
| | - John F Ouyang
- Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore, Singapore
| | - Fernando J Rossello
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
- Murdoch Children's Research Institute, Melbourne, Victoria, Australia
| | - Hun S Chy
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
- Biomedical Manufacturing, Commonwealth Scientific and Industrial Research Organisation, Melbourne, Victoria, Australia
| | - Owen J L Rackham
- Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore, Singapore
- School of Biological Sciences, University of Southampton, Southampton, UK
| | - Andrew L Laslett
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
- Biomedical Manufacturing, Commonwealth Scientific and Industrial Research Organisation, Melbourne, Victoria, Australia
| | - James Breen
- John Curtin School of Medical Research, College of Health and Medicine, Australian National University, Canberra, Australian Capital Territory, Australia
- South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia
| | - Geoffrey J Faulkner
- Mater Research Institute, University of Queensland, Brisbane, Queensland, Australia
- Queensland Brain Institute, University of Queensland, Brisbane, Queensland, Australia
| | - Christian M Nefzger
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia
- Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
| | - Jose M Polo
- Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia.
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia.
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia.
- Adelaide Centre for Epigenetics, School of Biomedicine, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, South Australia, Australia.
- The South Australian Immunogenomics Cancer Institute, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, South Australia, Australia.
| | - Ryan Lister
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia.
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia.
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12
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Saini P, Anugula S, Fong YW. The Role of ATP-Binding Cassette Proteins in Stem Cell Pluripotency. Biomedicines 2023; 11:1868. [PMID: 37509507 PMCID: PMC10377311 DOI: 10.3390/biomedicines11071868] [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: 05/17/2023] [Revised: 06/20/2023] [Accepted: 06/22/2023] [Indexed: 07/30/2023] Open
Abstract
Pluripotent stem cells (PSCs) are highly proliferative cells that can self-renew indefinitely in vitro. Upon receiving appropriate signals, PSCs undergo differentiation and can generate every cell type in the body. These unique properties of PSCs require specific gene expression patterns that define stem cell identity and dynamic regulation of intracellular metabolism to support cell growth and cell fate transitions. PSCs are prone to DNA damage due to elevated replicative and transcriptional stress. Therefore, mechanisms to prevent deleterious mutations in PSCs that compromise stem cell function or increase the risk of tumor formation from becoming amplified and propagated to progenitor cells are essential for embryonic development and for using PSCs including induced PSCs (iPSCs) as a cell source for regenerative medicine. In this review, we discuss the role of the ATP-binding cassette (ABC) superfamily in maintaining PSC homeostasis, and propose how their activities can influence cellular signaling and stem cell fate decisions. Finally, we highlight recent discoveries that not all ABC family members perform only canonical metabolite and peptide transport functions in PSCs; rather, they can participate in diverse cellular processes from genome surveillance to gene transcription and mRNA translation, which are likely to maintain the pristine state of PSCs.
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Affiliation(s)
- Prince Saini
- Brigham Regenerative Medicine Center, Brigham and Women’s Hospital, Boston, MA 02115, USA; (P.S.); (S.A.)
- Department of Medicine, Cardiovascular Medicine Division, Harvard Medical School, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Cambridge, MA 02138, USA
| | - Sharath Anugula
- Brigham Regenerative Medicine Center, Brigham and Women’s Hospital, Boston, MA 02115, USA; (P.S.); (S.A.)
- Department of Medicine, Cardiovascular Medicine Division, Harvard Medical School, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Cambridge, MA 02138, USA
| | - Yick W. Fong
- Brigham Regenerative Medicine Center, Brigham and Women’s Hospital, Boston, MA 02115, USA; (P.S.); (S.A.)
- Department of Medicine, Cardiovascular Medicine Division, Harvard Medical School, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Cambridge, MA 02138, USA
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13
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Edwards MM, Wang N, Massey DJ, Egli D, Koren A. Incomplete Reprogramming of DNA Replication Timing in Induced Pluripotent Stem Cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.12.544654. [PMID: 37398435 PMCID: PMC10312660 DOI: 10.1101/2023.06.12.544654] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/04/2023]
Abstract
Induced pluripotent stem cells (iPSC) are a widely used cell system and a foundation for cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which have the potential to affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing - a process linked to both genome regulation and genome stability - is efficiently reprogrammed to the embryonic state. To answer this, we profiled and compared genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicated their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibit delayed replication at heterochromatic regions containing genes downregulated in iPSC with incompletely reprogrammed DNA methylation. DNA replication delays were not the result of gene expression and DNA methylation aberrations and persisted after differentiating cells to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and lead to undesirable phenotypes in iPSCs, establishing it as an important genomic feature to consider when evaluating iPSC lines.
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Affiliation(s)
- Matthew M. Edwards
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
| | - Ning Wang
- Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, New York 10032, USA
- Columbia University Stem Cell Initiative, New York, New York 10032, USA
| | - Dashiell J. Massey
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
| | - Dieter Egli
- Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, New York 10032, USA
- Columbia University Stem Cell Initiative, New York, New York 10032, USA
| | - Amnon Koren
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
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14
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Kim GH, Kim J, Lee J, Jang DH. A novel pathogenic variant of DNMT3A associated with craniosynostosis: a case report of Heyn-Sproul-Jackson syndrome. Front Pediatr 2023; 11:1165638. [PMID: 37303757 PMCID: PMC10248406 DOI: 10.3389/fped.2023.1165638] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Accepted: 05/09/2023] [Indexed: 06/13/2023] Open
Abstract
Pathogenic variants of DNMT3A have been implicated in Tatton-Brown-Rahman syndrome, an overgrowth disorder with macrocephaly and intellectual disability. However, there are recent reports of variants in the same gene giving rise to an opposing clinical phenotype presenting with microcephaly, growth failure, and impaired development-named Heyn-Sproul-Jackson syndrome (HESJAS). Here, we present a case of HESJAS caused by a novel pathogenic variant of DNMT3A. A five-year-old girl presented with severe developmental delay. Perinatal and family history were non-contributory. Physical exam showed microcephaly and facial dysmorphic features, and neurodevelopmental assessments revealed profound global developmental delay. Brain magnetic resonance imaging findings were normal; however, brain 3D computed tomography revealed craniosynostosis. Next generation sequencing revealed a novel heterozygous variant in DNMT3A (NM_175629.2: c.1012_1014 + 3del). The patient's parents did not carry the variant. In this report, a novel feature associated with HESJAS (craniosynostosis) is described, along with a more detailed account of clinical manifestations than those in the original report.
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Affiliation(s)
- Ga Hye Kim
- Department of Rehabilitation Medicine, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Jaewon Kim
- Department of Rehabilitation Medicine, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Jaewoong Lee
- Department of Laboratory Medicine, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Dae-Hyun Jang
- Department of Rehabilitation Medicine, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
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15
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Kang H, Hasselbeck S, Taškova K, Wang N, Oosten LNV, Mrowka R, Utikal J, Andrade-Navarro MA, Wang J, Wölfl S, Cheng X. Development of a next-generation endogenous OCT4 inducer and its anti-aging effect in vivo. Eur J Med Chem 2023; 257:115513. [PMID: 37253308 DOI: 10.1016/j.ejmech.2023.115513] [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: 03/24/2023] [Revised: 05/05/2023] [Accepted: 05/23/2023] [Indexed: 06/01/2023]
Abstract
The identification of small molecules capable of replacing transcription factors has been a longstanding challenge in the generation of human chemically induced pluripotent stem cells (iPSCs). Recent studies have shown that ectopic expression of OCT4, one of the master pluripotency regulators, compromised the developmental potential of resulting iPSCs, This highlights the importance of finding endogenous OCT4 inducers for the generation of clinical-grade human iPSCs. Through a cell-based high throughput screen, we have discovered several new OCT4-inducing compounds (O4Is). In this work, we prepared metabolically stable analogues, including O4I4, which activate endogenous OCT4 and associated signaling pathways in various cell lines. By combining these with a transcription factor cocktail consisting of SOX2, KLF4, MYC, and LIN28 (referred to as "CSKML") we achieved to reprogram human fibroblasts into a stable and authentic pluripotent state without the need for exogenous OCT4. In Caenorhabditis elegans and Drosophila, O4I4 extends lifespan, suggesting the potential application of OCT4-inducing compounds in regenerative medicine and rejuvenation therapy.
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Affiliation(s)
- Han Kang
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany
| | - Sebastian Hasselbeck
- Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt am Main, Germany
| | - Katerina Taškova
- Faculty of Biology, Johannes Gutenberg University Mainz, Germany
| | - Nessa Wang
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany
| | - Luuk N van Oosten
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany
| | - Ralf Mrowka
- Experimentelle Nephrologie, KIM III, Universitätsklinikum, Jena, Germany
| | - Jochen Utikal
- Skin Cancer Unit (G300), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | | | - Jichang Wang
- Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Stefan Wölfl
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany
| | - Xinlai Cheng
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt am Main, Germany; Frankfurt Cancer Institute, Germany.
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16
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Poondi Krishnan V, Morone B, Toubiana S, Krzak M, Fioriniello S, Della Ragione F, Strazzullo M, Angelini C, Selig S, Matarazzo MR. The aberrant epigenome of DNMT3B-mutated ICF1 patient iPSCs is amenable to correction, with the exception of a subset of regions with H3K4me3- and/or CTCF-based epigenetic memory. Genome Res 2023; 33:169-183. [PMID: 36828588 PMCID: PMC10069469 DOI: 10.1101/gr.276986.122] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Accepted: 01/12/2023] [Indexed: 02/26/2023]
Abstract
Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to immunodeficiency, centromeric instability, facial anomalies syndrome, type 1 (ICF1). Although several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-derived induced pluripotent stem cells (iPSCs) and their CRISPR-Cas9-corrected clones to determine whether DNMT3B correction can globally overcome DNA methylation defects and related changes in the epigenome. Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1 patient peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are premarked during early development. CRISPR-corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions reacquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increases in H3K4me3 levels and/or abnormal CTCF binding, the epigenetic memory persists, and hypomethylation remains uncorrected. Overall, we demonstrate that restoring the catalytic activity of DNMT3B can reverse the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this rescue, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.
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Affiliation(s)
- Varsha Poondi Krishnan
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy
| | - Barbara Morone
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy
| | - Shir Toubiana
- Department of Genetics and Developmental Biology, Rappaport Faculty of Medicine and Research Institute, Technion, Haifa 31096, Israel
| | - Monika Krzak
- Institute for Applied Computing (IAC) "Mauro Picone", CNR, Naples 80131 Italy
| | - Salvatore Fioriniello
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy
| | - Floriana Della Ragione
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy.,IRCCS Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Isernia 86077, Italy
| | - Maria Strazzullo
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy;
| | - Claudia Angelini
- Institute for Applied Computing (IAC) "Mauro Picone", CNR, Naples 80131 Italy;
| | - Sara Selig
- Department of Genetics and Developmental Biology, Rappaport Faculty of Medicine and Research Institute, Technion, Haifa 31096, Israel; .,Laboratory of Molecular Medicine, Rambam Health Care Campus, Haifa 31096, Israel
| | - Maria R Matarazzo
- Institute of Genetics and Biophysics Adriano Buzzati Traverso, (IGB-ABT) CNR, Naples 80131, Italy
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17
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Galiakberova AA, Brovkina OI, Kondratyev NV, Artyuhov AS, Momotyuk ED, Kulmukhametova ON, Lagunin AA, Shilov BV, Zadorozhny AD, Zakharov IS, Okorokova LS, Golimbet VE, Dashinimaev EB. Different iPSC-derived neural stem cells shows various spectrums of spontaneous differentiation during long term cultivation. Front Mol Neurosci 2023; 16:1037902. [PMID: 37201156 PMCID: PMC10186475 DOI: 10.3389/fnmol.2023.1037902] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2022] [Accepted: 03/23/2023] [Indexed: 05/20/2023] Open
Abstract
Introduction Culturing of human neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSC) is a promising area of research, as these cells have the potential to treat a wide range of neurological, neurodegenerative and psychiatric diseases. However, the development of optimal protocols for the production and long-term culturing of NSCs remains a challenge. One of the most important aspects of this problem is to determine the stability of NSCs during long-term in vitro passaging. To address this problem, our study was aimed at investigating the spontaneous differentiation profile in different iPSC-derived human NSCs cultures during long-term cultivation using. Methods Four different IPSC lines were used to generate NSC and spontaneously differentiated neural cultures using DUAL SMAD inhibition. These cells were analyzed at different passages using immunocytochemistry, qPCR, bulk transcriptomes and scRNA-seq. Results We found that various NSC lines generate significantly different spectrums of differentiated neural cells, which can also change significantly during long-term cultivation in vitro. Discussion Our results indicate that both internal (genetic and epigenetic) and external (conditions and duration of cultivation) factors influence the stability of NSCs. These results have important implications for the development of optimal NSCs culturing protocols and highlight the need to further investigate the factors influencing the stability of these cells in vitro.
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Affiliation(s)
- Adelya Albertovna Galiakberova
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
- Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Olga Igorevna Brovkina
- Federal Research and Clinical Center, Federal Medical-Biological Agency of Russia, Moscow, Russia
| | | | - Alexander Sergeevich Artyuhov
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
| | - Ekaterina Dmitrievna Momotyuk
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
| | | | - Alexey Aleksandrovich Lagunin
- Pirogov Russian National Research Medical University, Moscow, Russia
- Department of Bioinformatics, Institute of Biomedical Chemistry, Moscow, Russia
| | | | | | - Igor Sergeevitch Zakharov
- Department of Bioinformatics, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia
| | | | | | - Erdem Bairovich Dashinimaev
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
- Department of Bioinformatics, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia
- Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russia
- *Correspondence: Erdem Bairovich Dashinimaev,
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18
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Goissis MD, Cibelli JB. Early Cell Specification in Mammalian Fertilized and Somatic Cell Nuclear Transfer Embryos. Methods Mol Biol 2023; 2647:59-81. [PMID: 37041329 DOI: 10.1007/978-1-0716-3064-8_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/13/2023]
Abstract
Early cell specification in mammalian preimplantation embryos is an intricate cellular process that leads to coordinated spatial and temporal expression of specific genes. Proper segregation into the first two cell lineages, the inner cell mass (ICM) and the trophectoderm (TE), is imperative for developing the embryo proper and the placenta, respectively. Somatic cell nuclear transfer (SCNT) allows the formation of a blastocyst containing both ICM and TE from a differentiated cell nucleus, which means that this differentiated genome must be reprogrammed to a totipotent state. Although blastocysts can be generated efficiently through SCNT, the full-term development of SCNT embryos is impaired mostly due to placental defects. In this review, we examine the early cell fate decisions in fertilized embryos and compare them to observations in SCNT-derived embryos, in order to understand if these processes are affected by SCNT and could be responsible for the low success of reproductive cloning.
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Affiliation(s)
- Marcelo D Goissis
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.
| | - Jose B Cibelli
- Department of Animal Science, Michigan State University, East Lansing, MI, USA
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19
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Maksimova VP, Usalka OG, Makus YV, Popova VG, Trapeznikova ES, Khayrieva GI, Sagitova GR, Zhidkova EM, Prus AY, Yakubovskaya MG, Kirsanov KI. Aberrations of DNA methylation in cancer. ADVANCES IN MOLECULAR ONCOLOGY 2022. [DOI: 10.17650/2313-805x-2022-9-4-24-40] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
DNA methylation is a chromatin modification that plays an important role in the epigenetic regulation of gene expression. Changes in DNA methylation patterns are characteristic of many malignant neoplasms. DNA methylation is occurred by DNA methyltransferases (DNMTs), while demethylation is mediated by TET family proteins. Mutations and changes in the expression profile of these enzymes lead to DNA hypo- and hypermethylation and have a strong impact on carcinogenesis. In this review, we considered the key aspects of the mechanisms of regulation of DNA methylation and demethylation, and also analyzed the role of DNA methyltransferases and TET family proteins in the pathogenesis of various malignant neoplasms.During the preparation of the review, we used the following biomedical literature information bases: Scopus (504), PubMed (553), Web of Science (1568), eLibrary (190). To obtain full-text documents, the electronic resources of PubMed Central (PMC), Science Direct, Research Gate, CyberLeninka were used. To analyze the mutational profile of epigenetic regulatory enzymes, we used the cBioportal portal (https://www.cbioportal.org / ), data from The AACR Project GENIE Consortium (https://www.mycancergenome.org / ), COSMIC, Clinvar, and The Cancer Genome Atlas (TCGA).
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Affiliation(s)
- V. P. Maksimova
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia
| | - O. G. Usalka
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; Sechenov First Moscow State Medical University, Ministry of Health of Russia
| | - Yu. V. Makus
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; Peoples’ Friendship University of Russia
| | - V. G. Popova
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; Mendeleev University of Chemical Technology of Russia
| | - E. S. Trapeznikova
- Sechenov First Moscow State Medical University, Ministry of Health of Russia
| | - G. I. Khayrieva
- Sechenov First Moscow State Medical University, Ministry of Health of Russia
| | - G. R. Sagitova
- Sechenov First Moscow State Medical University, Ministry of Health of Russia
| | - E. M. Zhidkova
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia
| | - A. Yu. Prus
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; MIREA – Russian Technological University
| | - M. G. Yakubovskaya
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia
| | - K. I. Kirsanov
- N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; Peoples’ Friendship University of Russia
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20
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Andrews PW, Barbaric I, Benvenisty N, Draper JS, Ludwig T, Merkle FT, Sato Y, Spits C, Stacey GN, Wang H, Pera MF. The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells. Cell Stem Cell 2022; 29:1624-1636. [PMID: 36459966 DOI: 10.1016/j.stem.2022.11.006] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 11/08/2022] [Accepted: 11/08/2022] [Indexed: 12/05/2022]
Abstract
It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.
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Affiliation(s)
- Peter W Andrews
- Centre for Stem Cell Biology, School of Biological Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK; Steering Committee, International Stem Cell Initiative
| | - Ivana Barbaric
- Centre for Stem Cell Biology, School of Biological Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK; Steering Committee, International Stem Cell Initiative
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel; Steering Committee, International Stem Cell Initiative
| | - Jonathan S Draper
- Stem Cell Network, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada; Steering Committee, International Stem Cell Initiative
| | - Tenneille Ludwig
- WiCell Research Institute, Madison, WI, USA; University of Wisconsin-Madison, Madison, WI 53719, USA; Steering Committee, International Stem Cell Initiative
| | - Florian T Merkle
- Wellcome Trust-Medical Research Council Institute of Metabolic Science, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0QQ, UK; Steering Committee, International Stem Cell Initiative
| | - Yoji Sato
- Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa 210-9501, Japan; Steering Committee, International Stem Cell Initiative
| | - Claudia Spits
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium; Steering Committee, International Stem Cell Initiative
| | - Glyn N Stacey
- International Stem Cell Banking Initiative, 2 High Street, Barley, UK; National Stem Cell Resource Centre, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100190, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China; Steering Committee, International Stem Cell Initiative
| | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101, Beijing, China; Beijing Institute for Stem Cell and Regenerative Medicine, 100101, Beijing, China; Steering Committee, International Stem Cell Initiative
| | - Martin F Pera
- The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA; Steering Committee, International Stem Cell Initiative.
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21
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Navarro M, Halstead MM, Rincon G, Mutto AA, Ross PJ. bESC from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells. Reproduction 2022; 164:243-257. [PMID: 35951478 DOI: 10.1530/rep-22-0063] [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: 02/16/2022] [Accepted: 08/11/2022] [Indexed: 11/08/2022]
Abstract
In brief Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos. Abstract Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.
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Affiliation(s)
- M Navarro
- Instituto de Investigaciones Biotecnológicas 'Dr Rodolfo Ugalde', UNSAM-CONICET, Buenos Aires, Argentina
- Department of Animal Science, University of California, Davis, California, USA
| | - M M Halstead
- Department of Animal Science, University of California, Davis, California, USA
| | | | - A A Mutto
- Instituto de Investigaciones Biotecnológicas 'Dr Rodolfo Ugalde', UNSAM-CONICET, Buenos Aires, Argentina
| | - P J Ross
- Department of Animal Science, University of California, Davis, California, USA
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22
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Arez M, Eckersley-Maslin M, Klobučar T, von Gilsa Lopes J, Krueger F, Mupo A, Raposo AC, Oxley D, Mancino S, Gendrel AV, Bernardes de Jesus B, da Rocha ST. Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation. Nat Commun 2022; 13:5432. [PMID: 36114205 PMCID: PMC9481624 DOI: 10.1038/s41467-022-33013-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Accepted: 08/29/2022] [Indexed: 11/24/2022] Open
Abstract
Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.
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Affiliation(s)
- Maria Arez
- iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
| | - Melanie Eckersley-Maslin
- Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, United Kingdom
- Peter MacCallum Cancer Centre, Melbourne, Victoria, 3000, Australia
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Victoria, 3010, Australia
- Department of Anatomy and Physiology, The University of Melbourne, Victoria, 3010, Australia
| | - Tajda Klobučar
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
- National Institute of Chemistry, Ljubljana, Slovenia
| | - João von Gilsa Lopes
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
- NOVA Medical School|Faculdade de Ciências Médicas, NMS|FCM, Universidade Nova de Lisboa, Lisboa, Portugal
| | - Felix Krueger
- Bioinformatics Group, Babraham Institute, Cambridge, CB22 3AT, United Kingdom
- Altos Labs, Cambridge, United Kingdom
| | - Annalisa Mupo
- Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, United Kingdom
- Altos Labs, Cambridge, United Kingdom
| | - Ana Cláudia Raposo
- iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
| | - David Oxley
- Mass Spectrometry Facility, The Babraham Institute, Cambridge, United Kingdom
| | - Samantha Mancino
- iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
| | - Anne-Valerie Gendrel
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
- Genetics and Developmental Biology Unit, Institut Curie, INSERM U934, CNRS UMR3215, PSL University, Paris, France
| | - Bruno Bernardes de Jesus
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
- Department of Medical Sciences and Institute of Biomedicine - iBiMED, University of Aveiro, 3810-193, Aveiro, Portugal
| | - Simão Teixeira da Rocha
- iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
- Associate Laboratory i4HB Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.
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Li Y, Sun Q. Epigenetic manipulation to improve mouse SCNT embryonic development. Front Genet 2022; 13:932867. [PMID: 36110221 PMCID: PMC9468881 DOI: 10.3389/fgene.2022.932867] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Accepted: 07/29/2022] [Indexed: 11/29/2022] Open
Abstract
Cloned mammals can be achieved through somatic cell nuclear transfer (SCNT), which involves reprogramming of differentiated somatic cells into a totipotent state. However, low cloning efficiency hampers its application severely. Cloned embryos have the same DNA as donor somatic cells. Therefore, incomplete epigenetic reprogramming accounts for low development of cloned embryos. In this review, we describe recent epigenetic barriers in SCNT embryos and strategies to correct these epigenetic defects and avoid the occurrence of abnormalities in cloned animals.
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Affiliation(s)
- Yamei Li
- University of Chinese Academy of Sciences, Beijing, China
- Institute of Neuroscience, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China
| | - Qiang Sun
- University of Chinese Academy of Sciences, Beijing, China
- Institute of Neuroscience, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China
- *Correspondence: Qiang Sun,
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24
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Gullapalli VK, Zarbin MA. New Prospects for Retinal Pigment Epithelium Transplantation. Asia Pac J Ophthalmol (Phila) 2022; 11:302-313. [PMID: 36041145 DOI: 10.1097/apo.0000000000000521] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 02/28/2022] [Indexed: 11/26/2022] Open
Abstract
ABSTRACT Retinal pigment epithelium (RPE) transplants rescue photoreceptors in selected animal models of retinal degenerative disease. Early clinical studies of RPE transplants as treatment for age-related macular degeneration (AMD) included autologous and allogeneic transplants of RPE suspensions and RPE sheets for atrophic and neovascular complications of AMD. Subsequent studies explored autologous RPE-Bruch membrane-choroid transplants in patients with neovascular AMD with occasional marked visual benefit, which establishes a rationale for RPE transplants in late-stage AMD. More recent work has involved transplantation of autologous and allogeneic stem cell-derived RPE for patients with AMD and those with Stargardt disease. These early-stage clinical trials have employed RPE suspensions and RPE monolayers on biocompatible scaffolds. Safety has been well documented, but evidence of efficacy is variable. Current research involves development of better scaffolds, improved modulation of immune surveillance, and modification of the extracellular milieu to improve RPE survival and integration with host retina.
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Affiliation(s)
| | - Marco A Zarbin
- Iinstitute of Ophthalmology and visual Science, Rutgers-New Jersey Medical School, Rutgers University, Newark, NJ, US
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25
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Haraguchi D, Nakamura T. Pramef12 enhances reprogramming into naïve iPS cells. Biochem Biophys Rep 2022; 30:101267. [PMID: 35592616 PMCID: PMC9111934 DOI: 10.1016/j.bbrep.2022.101267] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2022] [Revised: 04/07/2022] [Accepted: 04/20/2022] [Indexed: 11/22/2022] Open
Abstract
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Somatic cell nuclear transfer can also be utilized to reprogram somatic cells into totipotent embryos, suggesting that factors present in oocytes potentially enhance the efficiency of iPS cell generation. Here, we showed that preferentially expressed antigen of melanoma family member 12 (Pramef12), which is highly expressed in oocytes, enhances the generation of iPS cells from mouse fibroblasts. Overexpression of Pramef12 during the early phase of OKSM-induced reprogramming enhanced the efficiency of iPS cell derivation. In addition, overexpression of Pramef12 also enhanced expression of naïve pluripotency-associated genes, Gtl2 located within the Dlk1–Dio3 imprinted region essential for full pluripotency, glycolysis-associated genes, and oxidative phosphorylation-associated genes, and it promoted mesenchymal-to-epithelial transition during iPS cell generation. Furthermore, Pramef12 greatly activated β-catenin during iPS cell generation. These observations suggested that Pramef12 enhances OKSM-induced reprogramming via activation of the Wnt/β-catenin pathway.
Pramef12 enhances OKSM-induced reprogramming into naïve iPS cells. Pramef12 enhances expression of naïve pluripotency-associated genes, essential genes for full pluripotency, glycolysis-associated genes, and oxidative phosphorylation-associated genes. Pramef12 promotes mesenchymal-to-epithelial transition during iPS cell generation. Pramef12 enhances OKSM-induced reprogramming via activation of the Wnt/β-catenin pathway.
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Affiliation(s)
| | - Toshinobu Nakamura
- Gaduate School of Bio-Science, Japan
- Department of Bio-Science, Japan
- Genome Editing Research Institute, Ngahama Institute of Bio-Science and Technology, Shiga, 526-0829, Japan
- Corresponding author. Laboratory for epigenetic regulation, Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, Japan.
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26
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Li J, Pinto-Duarte A, Zander M, Cuoco MS, Lai CY, Osteen J, Fang L, Luo C, Lucero JD, Gomez-Castanon R, Nery JR, Silva-Garcia I, Pang Y, Sejnowski TJ, Powell SB, Ecker JR, Mukamel EA, Behrens MM. Dnmt3a knockout in excitatory neurons impairs postnatal synapse maturation and increases the repressive histone modification H3K27me3. eLife 2022; 11:e66909. [PMID: 35604009 PMCID: PMC9170249 DOI: 10.7554/elife.66909] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Accepted: 05/22/2022] [Indexed: 11/13/2022] Open
Abstract
Two epigenetic pathways of transcriptional repression, DNA methylation and polycomb repressive complex 2 (PRC2), are known to regulate neuronal development and function. However, their respective contributions to brain maturation are unknown. We found that conditional loss of the de novo DNA methyltransferase Dnmt3a in mouse excitatory neurons altered expression of synapse-related genes, stunted synapse maturation, and impaired working memory and social interest. At the genomic level, loss of Dnmt3a abolished postnatal accumulation of CG and non-CG DNA methylation, leaving adult neurons with an unmethylated, fetal-like epigenomic pattern at ~222,000 genomic regions. The PRC2-associated histone modification, H3K27me3, increased at many of these sites. Our data support a dynamic interaction between two fundamental modes of epigenetic repression during postnatal maturation of excitatory neurons, which together confer robustness on neuronal regulation.
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Affiliation(s)
- Junhao Li
- Department of Cognitive Science, University of California, San DiegoLa JollaUnited States
| | - Antonio Pinto-Duarte
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Mark Zander
- Genomic Analysis Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Michael S Cuoco
- Bioinformatics and Systems Biology Graduate Program, University of California, San DiegoLa JollaUnited States
| | - Chi-Yu Lai
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Julia Osteen
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Linjing Fang
- Waitt Advanced Biophotonics Core, Salk Institute for Biological StudiesLa JollaUnited States
| | - Chongyuan Luo
- Genomic Analysis Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
- Howard Hughes Medical Institute, Salk Institute for Biological StudiesLa JollaUnited States
| | - Jacinta D Lucero
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Rosa Gomez-Castanon
- Genomic Analysis Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Joseph R Nery
- Genomic Analysis Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
- Howard Hughes Medical Institute, Salk Institute for Biological StudiesLa JollaUnited States
| | - Isai Silva-Garcia
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Yan Pang
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Terrence J Sejnowski
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Susan B Powell
- Department of Psychiatry, University of California, San DiegoLa JollaUnited States
| | - Joseph R Ecker
- Genomic Analysis Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
| | - Eran A Mukamel
- Department of Cognitive Science, University of California, San DiegoLa JollaUnited States
| | - M Margarita Behrens
- Computational Neurobiology Laboratory, Salk Institute for Biological StudiesLa JollaUnited States
- Department of Psychiatry, University of California, San DiegoLa JollaUnited States
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27
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Harvey JP, Sladen PE, Yu-Wai-Man P, Cheetham ME. Induced Pluripotent Stem Cells for Inherited Optic Neuropathies-Disease Modeling and Therapeutic Development. J Neuroophthalmol 2022; 42:35-44. [PMID: 34629400 DOI: 10.1097/wno.0000000000001375] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Inherited optic neuropathies (IONs) cause progressive irreversible visual loss in children and young adults. There are limited disease-modifying treatments, and most patients progress to become severely visually impaired, fulfilling the legal criteria for blind registration. The seminal discovery of the technique for reprogramming somatic nondividing cells into induced pluripotent stem cells (iPSCs) has opened several exciting opportunities in the field of ION research and treatment. EVIDENCE ACQUISITION A systematic review of the literature was conducted with PubMed using the following search terms: autosomal dominant optic atrophy, ADOA, dominant optic atrophy, DOA, Leber hereditary optic neuropathy, LHON, optic atrophy, induced pluripotent stem cell, iPSC, iPSC derived, iPS, stem cell, retinal ganglion cell, and RGC. Clinical trials were identified on the ClinicalTrials.gov website. RESULTS This review article is focused on disease modeling and the therapeutic strategies being explored with iPSC technologies for the 2 most common IONs, namely, dominant optic atrophy and Leber hereditary optic neuropathy. The rationale and translational advances for cell-based and gene-based therapies are explored, as well as opportunities for neuroprotection and drug screening. CONCLUSIONS iPSCs offer an elegant, patient-focused solution to the investigation of the genetic defects and disease mechanisms underpinning IONs. Furthermore, this group of disorders is uniquely amenable to both the disease modeling capability and the therapeutic potential that iPSCs offer. This fast-moving area will remain at the forefront of both basic and translational ION research in the coming years, with the potential to accelerate the development of effective therapies for patients affected with these blinding diseases.
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Affiliation(s)
- Joshua Paul Harvey
- UCL Institute of Ophthalmology (JPH, PES, PY-W-M, MC), London, United Kingdom; Moorfields Eye Hospital NHS Foundation Trust (JPH, PY-W-M), London, United Kingdom; Department of Clinical Neurosciences (PY-W-M), Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom; and Department of Clinical Neurosciences (PY-W-M), John van Geest Centre for Brain Repair and MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
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28
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Martini P, Sales G, Diamante L, Perrera V, Colantuono C, Riccardo S, Cacchiarelli D, Romualdi C, Martello G. BrewerIX enables allelic expression analysis of imprinted and X-linked genes from bulk and single-cell transcriptomes. Commun Biol 2022; 5:146. [PMID: 35177756 PMCID: PMC8854590 DOI: 10.1038/s42003-022-03087-4] [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: 07/19/2021] [Accepted: 01/24/2022] [Indexed: 12/12/2022] Open
Abstract
Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed mono-allelically in order to fine-tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumours and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variants (SNVs) are readily detectable by RNA-sequencing allowing the determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about the allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyze both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data. BrewerIX confirmed previous observations regarding the bi-allelic expression of some imprinted genes in naive pluripotent cells and extended them to preimplantation embryos. BrewerIX also identified misregulated imprinted genes in breast cancer cells and in human organoids and identified genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer, iPSCs and organoids. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising the robustness and reproducibility of future studies.
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Affiliation(s)
- Paolo Martini
- Department of Biology, University of Padova, Padua, Italy
- Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
| | - Gabriele Sales
- Department of Biology, University of Padova, Padua, Italy
| | - Linda Diamante
- Department of Molecular Medicine, Medical School, University of Padova, Padua, Italy
| | - Valentina Perrera
- Department of Molecular Medicine, Medical School, University of Padova, Padua, Italy
- International School for Advanced Studies (SISSA/ISAS), Trieste, 34136, Italy
| | - Chiara Colantuono
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
| | - Sara Riccardo
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
| | - Davide Cacchiarelli
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- Department of Translational Medicine, University of Naples "Federico II", Naples, Italy
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29
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Rao I, Crisafulli L, Paulis M, Ficara F. Hematopoietic Cells from Pluripotent Stem Cells: Hope and Promise for the Treatment of Inherited Blood Disorders. Cells 2022; 11:cells11030557. [PMID: 35159366 PMCID: PMC8834203 DOI: 10.3390/cells11030557] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 01/28/2022] [Accepted: 02/03/2022] [Indexed: 01/26/2023] Open
Abstract
Inherited blood disorders comprise a large spectrum of diseases due to germline mutations in genes with key function in the hematopoietic system; they include immunodeficiencies, anemia or metabolic diseases. For most of them the only curative treatment is bone marrow transplantation, a procedure associated to severe complications; other therapies include red blood cell and platelet transfusions, which are dependent on donor availability. An alternative option is gene therapy, in which the wild-type form of the mutated gene is delivered into autologous hematopoietic stem cells using viral vectors. A more recent therapeutic perspective is gene correction through CRISPR/Cas9-mediated gene editing, that overcomes safety concerns due to insertional mutagenesis and allows correction of base substitutions in large size genes difficult to incorporate into vectors. However, applying this technique to genomic disorders caused by large gene deletions is challenging. Chromosomal transplantation has been proposed as a solution, using a universal source of wild-type chromosomes as donor, and induced pluripotent stem cells (iPSCs) as acceptor. One of the obstacles to be addressed for translating PSC research into clinical practice is the still unsatisfactory differentiation into transplantable hematopoietic stem or mature cells. We provide an overview of the recent progresses in this field and discuss challenges and potential of iPSC-based therapies for the treatment of inherited blood disorders.
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Affiliation(s)
- Ilaria Rao
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- Department of Biomedical Sciences, Humanitas University, Via Rita Levi Montalcini 4, 20090 Pieve Emanuele, Italy
| | - Laura Crisafulli
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
| | - Marianna Paulis
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
| | - Francesca Ficara
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
- Correspondence:
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30
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Yoshimatsu S, Nakajima M, Qian E, Sanosaka T, Sato T, Okano H. Homologous Recombination-Enhancing Factors Identified by Comparative Transcriptomic Analyses of Pluripotent Stem Cell of Human and Common Marmoset. Cells 2022; 11:cells11030360. [PMID: 35159172 PMCID: PMC8834151 DOI: 10.3390/cells11030360] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2021] [Revised: 01/12/2022] [Accepted: 01/19/2022] [Indexed: 02/04/2023] Open
Abstract
A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.
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Affiliation(s)
- Sho Yoshimatsu
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
- Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama 351-0198, Japan
| | - Mayutaka Nakajima
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
| | - Emi Qian
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
| | - Tsukasa Sanosaka
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
| | - Tsukika Sato
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; (S.Y.); (M.N.); (E.Q.); (T.S.); (T.S.)
- Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama 351-0198, Japan
- Correspondence:
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Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials. Cell Death Dis 2021; 12:1085. [PMID: 34785659 PMCID: PMC8595669 DOI: 10.1038/s41419-021-04384-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 10/26/2021] [Accepted: 10/29/2021] [Indexed: 11/08/2022]
Abstract
Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.
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32
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Inherent genomic properties underlie the epigenomic heterogeneity of human induced pluripotent stem cells. Cell Rep 2021; 37:109909. [PMID: 34731633 DOI: 10.1016/j.celrep.2021.109909] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Revised: 07/24/2021] [Accepted: 10/08/2021] [Indexed: 01/13/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) show variable differentiation potential due to their epigenomic heterogeneity, whose extent/attributes remain unclear, except for well-studied elements/chromosomes such as imprints and the X chromosomes. Here, we show that seven hiPSC lines with variable germline potential exhibit substantial epigenomic heterogeneity, despite their uniform transcriptomes. Nearly a quarter of autosomal regions bear potentially differential chromatin modifications, with promoters/CpG islands for H3K27me3/H2AK119ub1 and evolutionarily young retrotransposons for H3K4me3. We identify 145 large autosomal blocks (≥100 kb) with differential H3K9me3 enrichment, many of which are lamina-associated domains (LADs) in somatic but not in embryonic stem cells. A majority of these epigenomic heterogeneities are independent of genetic variations. We identify an X chromosome state with chromosome-wide H3K9me3 that stably prevents X chromosome erosion. Importantly, the germline potential of female hiPSCs correlates with X chromosome inactivation. We propose that inherent genomic properties, including CpG density, transposons, and LADs, engender epigenomic heterogeneity in hiPSCs.
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33
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Affiliation(s)
- Seungbok Yang
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea
| | - Yoonjae Cho
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea
| | - Jiwon Jang
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea
- Institute of Convergence Science, Yonsei University, Seoul 03722, Korea
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Horánszky A, Becker JL, Zana M, Ferguson-Smith AC, Dinnyés A. Epigenetic Mechanisms of ART-Related Imprinting Disorders: Lessons From iPSC and Mouse Models. Genes (Basel) 2021; 12:genes12111704. [PMID: 34828310 PMCID: PMC8620286 DOI: 10.3390/genes12111704] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Revised: 10/24/2021] [Accepted: 10/25/2021] [Indexed: 12/29/2022] Open
Abstract
The rising frequency of ART-conceived births is accompanied by the need for an improved understanding of the implications of ART on gametes and embryos. Increasing evidence from mouse models and human epidemiological data suggests that ART procedures may play a role in the pathophysiology of certain imprinting disorders (IDs), including Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Prader-Willi syndrome, and Angelman syndrome. The underlying molecular basis of this association, however, requires further elucidation. In this review, we discuss the epigenetic and imprinting alterations of in vivo mouse models and human iPSC models of ART. Mouse models have demonstrated aberrant regulation of imprinted genes involved with ART-related IDs. In the past decade, iPSC technology has provided a platform for patient-specific cellular models of culture-associated perturbed imprinting. However, despite ongoing efforts, a deeper understanding of the susceptibility of iPSCs to epigenetic perturbation is required if they are to be reliably used for modelling ART-associated IDs. Comparing the patterns of susceptibility of imprinted genes in mouse models and IPSCs in culture improves the current understanding of the underlying mechanisms of ART-linked IDs with implications for our understanding of the influence of environmental factors such as culture and hormone treatments on epigenetically important regions of the genome such as imprints.
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Affiliation(s)
- Alex Horánszky
- BioTalentum Ltd., H-2100 Gödöllő, Hungary; (A.H.); (M.Z.)
- Department of Physiology and Animal Health, Institute of Physiology and Animal Health, Hungarian University of Agriculture and Life Sciences, H-2100 Gödöllő, Hungary
| | - Jessica L. Becker
- Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK; (J.L.B.); (A.C.F.-S.)
| | - Melinda Zana
- BioTalentum Ltd., H-2100 Gödöllő, Hungary; (A.H.); (M.Z.)
| | - Anne C. Ferguson-Smith
- Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK; (J.L.B.); (A.C.F.-S.)
| | - András Dinnyés
- BioTalentum Ltd., H-2100 Gödöllő, Hungary; (A.H.); (M.Z.)
- Department of Physiology and Animal Health, Institute of Physiology and Animal Health, Hungarian University of Agriculture and Life Sciences, H-2100 Gödöllő, Hungary
- HCEMM-USZ Stem Cell Research Group, Hungarian Centre of Excellence for Molecular Medicine, H-6723 Szeged, Hungary
- Department of Cell Biology and Molecular Medicine, University of Szeged, H-6720 Szeged, Hungary
- Correspondence: ; Tel.: +36-20-510-9632; Fax: +36-28-526-151
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Keshet G, Benvenisty N. Large-scale analysis of imprinting in naive human pluripotent stem cells reveals recurrent aberrations and a potential link to FGF signaling. Stem Cell Reports 2021; 16:2520-2533. [PMID: 34597600 PMCID: PMC8514966 DOI: 10.1016/j.stemcr.2021.09.002] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Revised: 09/01/2021] [Accepted: 09/02/2021] [Indexed: 01/21/2023] Open
Abstract
Genomic imprinting is a parent-of-origin dependent monoallelic expression of genes. Previous studies showed that conversion of primed human pluripotent stem cells (hPSCs) into naive pluripotency is accompanied by genome-wide loss of methylation that includes imprinted loci. However, the extent of aberrant biallelic expression of imprinted genes is still unknown. Here, we analyze loss of imprinting (LOI) in a large cohort of both bulk and single-cell RNA sequencing samples of naive and primed hPSCs. We show that naive hPSCs exhibit high levels of non-random LOI, with bias toward paternally methylated imprinting control regions. Importantly, we show that different protocols used for the primed to naive conversion led to different extents of LOI, tightly correlated to FGF signaling. This analysis sheds light on the process of LOI occurring during the conversion to naive pluripotency and highlights the importance of these events when modeling disease and development or when utilizing the cells for therapy.
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Affiliation(s)
- Gal Keshet
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel.
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Capparè P, Tetè G, Sberna MT, Panina-Bordignon P. The Emerging Role of Stem Cells in Regenerative Dentistry. Curr Gene Ther 2021; 20:259-268. [PMID: 32811413 DOI: 10.2174/1566523220999200818115803] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 07/25/2020] [Accepted: 07/29/2020] [Indexed: 02/06/2023]
Abstract
Progress of modern dentistry is accelerating at a spectacular speed in the scientific, technological and clinical areas. Practical examples are the advancement in the digital field, which has guaranteed an average level of prosthetic practices for all patients, as well as other scientific developments, including research on stem cell biology. Given their plasticity, defined as the ability to differentiate into specific cell lineages with a capacity of almost unlimited self-renewal and release of trophic/immunomodulatory factors, stem cells have gained significant scientific and commercial interest in the last 15 years. Stem cells that can be isolated from various tissues of the oral cavity have emerged as attractive sources for bone and dental regeneration, mainly due to their ease of accessibility. This review will present the current understanding of emerging conceptual and technological issues of the use of stem cells to treat bone and dental loss defects. In particular, we will focus on the clinical application of stem cells, either directly isolated from oral sources or in vitro reprogrammed from somatic cells (induced pluripotent stem cells). Research aimed at further unraveling stem cell plasticity will allow to identify optimal stem cell sources and characteristics, to develop novel regenerative tools in dentistry.
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Affiliation(s)
- Paolo Capparè
- Department of Dentistry, IRCCS San Raffaele Hospital, Milan, Italy,Dental School, Vita-Salute San Raffaele University, School of Medicine, Milan, Italy
| | - Giulia Tetè
- Department of Dentistry, IRCCS San Raffaele Hospital, Milan, Italy
| | | | - Paola Panina-Bordignon
- Neuroimmunology Unit, Institute of Experimental Neurology, IRCCS San Raffaele Hospital, Milan, Italy,Dental School, Vita-Salute San Raffaele University, School of Medicine, Milan, Italy
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37
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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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Wang X, Zhao J, Zhu Z. Exogenous Expression of pou5f3 Facilitates the Development of Somatic Cell Nuclear Transfer Embryos in Zebrafish at the Early Stage. Cell Reprogram 2021; 23:191-197. [PMID: 34101505 DOI: 10.1089/cell.2021.0013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Enucleated oocytes can reprogram differentiated nuclei to totipotency after somatic cell nuclear transfer (SCNT), which is valuable in understanding nuclear reprogramming and generating genetically modified animals. To date, reprogramming efficiency is low and the development of SCNT embryos is not going as well as anticipated. To further disclose the reprogramming mechanisms during SCNT zebrafish embryo development, we examined the expression patterns of transcription regulation factors and regulated them by mRNA and morpholino microinjection. In this study, we show that stem cell-related transcription factors are downregulated in zebrafish SCNT embryos at the blastula stage. Exogenous expression of pou5f3 at the single-cell stage improves SCNT embryo development from the blastula to the gastrula stage. We also found that exogenous expression of klf4 or sox2 decreases SCNT embryo development from the blastula to the gastrula stage, while expression of nanog is necessary for the development of SCNT embryos. Our results conclude that zebrafish pou5f3 facilitates the development of SCNT embryos from the blastula to gastrula stage.
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Affiliation(s)
- Xuegeng Wang
- College of Life Sciences, Peking University, Beijing, P.R. China.,Institute of Modern Aquaculture Science and Engineering, College of Life Sciences, South China Normal University, Guangzhou, P.R. China
| | - Jue Zhao
- College of Life Sciences, Peking University, Beijing, P.R. China
| | - Zuoyan Zhu
- College of Life Sciences, Peking University, Beijing, P.R. China
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Retention of Somatic Memory Associated with Cell Identity, Age and Metabolism in Induced Pluripotent Stem (iPS) Cells Reprogramming. Stem Cell Rev Rep 2021; 16:251-261. [PMID: 32016780 DOI: 10.1007/s12015-020-09956-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The discovery of induced pluripotent stem (iPS) cells in 2006 marked a major breakthrough in regenerative medicine, enabling reversal of terminally differentiated somatic cells into pluripotent stem cells. The embryonic stem (ES) cells-like pluripotency and unlimited self-renewal capability of iPS cells have granted them enormous potential in many applications, particularly regenerative therapy. Unlike ES cells, however, iPS cells exhibit somatic memories which were carried over from the tissue of origin thus limited its translation in clinical applications. This review provides an updated overview of the retention of various somatic memories associated with the cellular identity, age and metabolism of tissue of origin in iPS cells. The influence of cell types, stage of maturation, age and various other factors on the retention of somatic memory has been discussed. Recent evidence of somatic memory in the form of epigenetic, transcriptomic, metabolic signatures and its functional manifestations in both in vitro and in vivo settings also have been reviewed. The increasing number of studies which had adopted isogenic cell lines for comparisons in recent years had facilitated the identification of genuine somatic memories. These memories functionally affect iPS cells and its derivatives and are potentially tumorigenic thus, raising concerns on their safety in clinical application. Various approaches for memory erasure had since being reported and their efficacies were highlighted in this review.
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40
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Yang Z, Zhao J, Li J, Wang J, Wang W. Genome-wide DNA methylation profiling of high-fat emulsion-induced hyperlipidemia mice intervened by a polysaccharide from Cyclocarya paliurus (Batal) Iljinskaja. Food Chem Toxicol 2021. [DOI: https://doi.org/10.1016/j.fct.2021.112230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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41
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Yang Z, Zhao J, Li J, Wang J, Wang W. Genome-wide DNA methylation profiling of high-fat emulsion-induced hyperlipidemia mice intervened by a polysaccharide from Cyclocarya paliurus (Batal) Iljinskaja. Food Chem Toxicol 2021; 152:112230. [PMID: 33878369 DOI: 10.1016/j.fct.2021.112230] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Revised: 04/09/2021] [Accepted: 04/14/2021] [Indexed: 02/07/2023]
Abstract
Genome-wide DNA methylation was used to study the lipid-lowering effect of Cyclocarya paliurus (Batal) Iljinskaja polysaccharide (CPP). The objective of this study was to investigate the hypolipidemic effects and the potential underlying mechanisms of action of CPP-2 in high-fat emulsion (HFE)-induced mice. The results showed that CPP-2 reduced the level of genome-wide DNA methylation in the liver of HFE-induced mice, which had a lipid-lowering effect by regulating the AMP-activated protein kinase (AMPK) signaling-, fatty acid metabolism-, fatty acid biosynthesis- and adipocytokine signaling pathways. A series of lipid metabolism genes were screened out by conjoint analysis of the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Hereafter, fatty acid synthase (FAS) and peroxisome proliferators-activated receptor α (PPARα) as target genes were selected to validate the accuracy of the results. The findings demonstrated that CPP-2 might be effective in lowering the lipid content, thereby protecting against HFE-induced hyperlipidemia.
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Affiliation(s)
- Zhanwei Yang
- Key Lab for Agro-product Processing and Quality Control of Nanchang City, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China; School of Food Sciences and Engineering, South China University of Technology, Guangzhou, 510640, China
| | - Jing Zhao
- Guang' an Vocation &Technical College, Guang' an 638000, China
| | - Jing'en Li
- Key Lab for Agro-product Processing and Quality Control of Nanchang City, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China
| | - Jin Wang
- The State Centre of Quality Supervision and Inspection for Camellia Products (Jiangxi), Ganzhou 341000, China
| | - Wenjun Wang
- Key Lab for Agro-product Processing and Quality Control of Nanchang City, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
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42
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Lee AR, Park JH, Shim SH, Hong K, La H, Park KS, Lee DR. Genome stabilization by RAD51-stimulatory compound 1 enhances efficiency of somatic cell nuclear transfer-mediated reprogramming and full-term development of cloned mouse embryos. Cell Prolif 2021; 54:e13059. [PMID: 34021643 PMCID: PMC8249786 DOI: 10.1111/cpr.13059] [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: 02/22/2021] [Revised: 04/24/2021] [Accepted: 05/04/2021] [Indexed: 12/11/2022] Open
Abstract
OBJECTIVES The genetic instability and DNA damage arise during transcription factor-mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)-mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos. MATERIALS AND METHODS Whether the repair of genetic instability or double-strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT-derived embryos. RESULTS Here, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS-1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single-cell RNA-seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT-2-cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming-resistance regions. CONCLUSIONS This technology can contribute to the production of comparable cell sources for regenerative medicine.
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Affiliation(s)
- Ah Reum Lee
- Department of Biomedical Science, CHA University, Seongnam, Gyunggi-do, Korea.,CHA Advanced Research Institute, CHA University, Seongnam, Gyunggi-do, Korea
| | - Ji-Hoon Park
- Department of Biomedical Science, CHA University, Seongnam, Gyunggi-do, Korea
| | - Sung Han Shim
- Department of Biomedical Science, CHA University, Seongnam, Gyunggi-do, Korea
| | - Kwonho Hong
- Department of Stem Cell and Regenerative Biology, Konkuk University, Gwangjin-gu, Seoul, Korea
| | - Hyeonwoo La
- Department of Stem Cell and Regenerative Biology, Konkuk University, Gwangjin-gu, Seoul, Korea
| | - Kyung-Soon Park
- Department of Biomedical Science, CHA University, Seongnam, Gyunggi-do, Korea
| | - Dong Ryul Lee
- Department of Biomedical Science, CHA University, Seongnam, Gyunggi-do, Korea.,CHA Advanced Research Institute, CHA University, Seongnam, Gyunggi-do, Korea
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Kosanke M, Osetek K, Haase A, Wiehlmann L, Davenport C, Schwarzer A, Adams F, Kleppa MJ, Schambach A, Merkert S, Wunderlich S, Menke S, Dorda M, Martin U. Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes. Mol Ther 2021; 29:2535-2553. [PMID: 33831558 DOI: 10.1016/j.ymthe.2021.04.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Revised: 03/03/2021] [Accepted: 04/02/2021] [Indexed: 02/06/2023] Open
Abstract
Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.
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Affiliation(s)
- Maike Kosanke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Katarzyna Osetek
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Alexandra Haase
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Lutz Wiehlmann
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Colin Davenport
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Adrian Schwarzer
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Felix Adams
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Marc-Jens Kleppa
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Axel Schambach
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Sylvia Merkert
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Stephanie Wunderlich
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Sandra Menke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Marie Dorda
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany.
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Qiao S, Feng S, Wu Z, He T, Ma C, Peng Z, Tian E, Pan G. Functional Proliferating Human Hepatocytes: In Vitro Hepatocyte Model for Drug Metabolism, Excretion, and Toxicity. Drug Metab Dispos 2021; 49:305-313. [PMID: 33526515 DOI: 10.1124/dmd.120.000275] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
To develop a functional alternative hepatocyte model for primary human hepatocytes (PHHs) with proliferative property, essential drug metabolic, and transporter functions, proliferating human hepatocytes (ProliHHs) expanded from PHHs were fully characterized in vitro. Herein, ProliHHs generated from multiple PHHs donors could be expanded more than 200-fold within four passages and maintained their metabolic or transporter capacities partially. Furthermore, ProliHHs were able to regain the mature hepatic property after three-dimensional (3D) culture. Particularly, the downregulated mRNA expression and function of three major cytochrome P450 (P450) enzymes (CYP1A2, CYP2B6, and CYP3A4) in the proliferating process (ProliHHs-P) could be recovered by 3D culture. The metabolic variabilities across different PHHs donors could be inherited to their matured ProliHHs (ProliHHs-M). The intrinsic clearances of seven major P450 enzymes in ProliHHs-M correlated well (r = 0.87) with those in PHHs. Also, bile canaliculi structures could be observed in sandwich-cultured ProliHHs (SC-ProliHHs), and the biliary excretion index of four probe compounds [cholyl-lys-fluorescein, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (CDF), deuterium-labeled sodium taurocholate acid, and rosuvastatin] in SC-ProliHHs (>10%) were close to sandwich-cultured PHHs. More importantly, both ProliHHs-P and ProliHHs-M could be used to evaluate hepatotoxicity. Therefore, these findings demonstrated that the 3D and sandwich culture system could be used to recover the metabolic and transporter functions in ProliHHs for clearance prediction and cholestasis risk assessment, respectively. Together, ProliHHs could be a promising substitute for PHHs in drug metabolism, transport, and hepatotoxicity screening. SIGNIFICANCE STATEMENT: This report describes the study of drug metabolic capacities, efflux transporter functions, and toxicity assessments of proliferating human hepatocytes (ProliHHs). The metabolic variability in different primary human hepatocyte donors could be inherited by their matured ProliHHs derivatives. Also, ProliHHs could form canalicular networks in sandwich culture and display biliary excretion capacities. More importantly, both the proliferative and maturation statuses of ProliHHs could be used to evaluate hepatotoxicity. Together, ProliHHs were feasible to support drug candidate screening in hepatic metabolism, disposition, and toxicity.
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Affiliation(s)
- Shida Qiao
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Sisi Feng
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Zhitao Wu
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Ting He
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Chen Ma
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Zhaoliang Peng
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - E Tian
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
| | - Guoyu Pan
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
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Abstract
Somatic cell nuclear transfer (SCNT) is a powerful technique, although challenging, to study reprograming into the totipotent state of differentiated nuclei in mammals. This procedure was initially applied in farm animals, then rodents, and more recently in primates. Nuclear transfer of embryonic stem cells is known to be more efficient, but many types of somatic cells have now been successfully reprogramed with this procedure. Moreover, SCNT reprograming is more effective on a per cell basis than induced Pluripotent Stem Cells (iPSC) and provides interesting clues regarding the underlying processes. In this chapter, we describe the protocol of nuclear transfer in mouse that combines cell cycle synchronization of the donor cells, enucleation of metaphase II oocyte and Piezo-driven injection of a donor cell nucleus followed by activation of the reconstructed embryos and nonsurgical transfer into pseudo-pregnant mice. Moreover, this protocol includes two facultative steps to erase the epigenetic "memory" of the donor cells and improve chromatin remodeling by histones modifications targeting.
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Affiliation(s)
- Vincent Brochard
- Université Paris-Saclay, INRAE, ENVA, BREED U1198, Jouy-en-Josas, France
| | - Nathalie Beaujean
- Université Paris-Saclay, INRAE, ENVA, BREED U1198, Jouy-en-Josas, France. .,Univ Lyon, Université Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U1208, USC 1361, Bron, France.
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Bartoccetti M, van der Veer BK, Luo X, Khoueiry R, She P, Bajaj M, Xu J, Janiszewski A, Thienpont B, Pasque V, Koh KP. Regulatory Dynamics of Tet1 and Oct4 Resolve Stages of Global DNA Demethylation and Transcriptomic Changes in Reprogramming. Cell Rep 2021; 30:2150-2169.e9. [PMID: 32075734 DOI: 10.1016/j.celrep.2020.01.065] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2019] [Revised: 12/12/2019] [Accepted: 01/21/2020] [Indexed: 01/05/2023] Open
Abstract
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) involves the reactivation of endogenous pluripotency genes and global DNA demethylation, but temporal resolution of these events using existing markers is limited. Here, we generate murine transgenic lines harboring reporters for the 5-methylcytosine dioxygenase Tet1 and for Oct4. By monitoring dual reporter fluorescence during pluripotency entry, we identify a sequential order of Tet1 and Oct4 activation by proximal and distal regulatory elements. Full Tet1 activation marks an intermediate stage that accompanies predominantly repression of somatic genes, preceding full Oct4 activation, and distinguishes two waves of global DNA demethylation that target distinct genomic features but are uncoupled from transcriptional changes. Tet1 knockout shows that TET1 contributes to both waves of demethylation and activates germline regulatory genes in reprogramming intermediates but is dispensable for Oct4 reactivation. Our dual reporter system for time-resolving pluripotency entry thus refines the molecular roadmap of iPSC maturation.
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Affiliation(s)
- Michela Bartoccetti
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Bernard K van der Veer
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Xinlong Luo
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Rita Khoueiry
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Pinyi She
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Manmohan Bajaj
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Jiayi Xu
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Adrian Janiszewski
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Bernard Thienpont
- Department of Human Genetics, Laboratory for Functional Epigenetics, KU Leuven, 3000 Leuven, Belgium
| | - Vincent Pasque
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium
| | - Kian Peng Koh
- Department of Development and Regeneration, Stem Cell Institute Leuven, KU Leuven, 3000 Leuven, Belgium.
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hiPSCs for predictive modelling of neurodegenerative diseases: dreaming the possible. Nat Rev Neurol 2021; 17:381-392. [PMID: 33658662 PMCID: PMC7928200 DOI: 10.1038/s41582-021-00465-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/27/2021] [Indexed: 02/07/2023]
Abstract
Human induced pluripotent stem cells (hiPSCs) were first generated in 2007, but the full translational potential of this valuable tool has yet to be realized. The potential applications of hiPSCs are especially relevant to neurology, as brain cells from patients are rarely available for research. hiPSCs from individuals with neuropsychiatric or neurodegenerative diseases have facilitated biological and multi-omics studies as well as large-scale screening of chemical libraries. However, researchers are struggling to improve the scalability, reproducibility and quality of this descriptive disease modelling. Addressing these limitations will be the first step towards a new era in hiPSC research - that of predictive disease modelling - involving the correlation and integration of in vitro experimental data with longitudinal clinical data. This approach is a key element of the emerging precision medicine paradigm, in which hiPSCs could become a powerful diagnostic and prognostic tool. Here, we consider the steps necessary to achieve predictive modelling of neurodegenerative disease with hiPSCs, using Huntington disease as an example.
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48
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Zuo Y, Song M, Li H, Chen X, Cao P, Zheng L, Cao G. Analysis of the Epigenetic Signature of Cell Reprogramming by Computational DNA Methylation Profiles. Curr Bioinform 2020. [DOI: 10.2174/1574893614666190919103752] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Background:
DNA methylation plays an important role in the reprogramming process.
Understanding the underlying molecular mechanism of reprogramming is crucial for answering
fundamental questions regarding the transition of cell identity.
Methods:
In this study, based on the genome-wide DNA methylation data from different cell lines,
comparative methylation profiles were proposed to identify the epigenetic signature of cell
reprogramming.
Results:
The density profile of CpG methylation showed that pluripotent cells are more polarized
than Human Dermal Fibroblasts (HDF) cells. The heterogeneity of iPS has a greater deviation in
the DNA hypermethylation pattern. The result of regional distribution showed that the differential
CpG sites between pluripotent cells and HDFs tend to accumulate in the gene body and CpG shelf
regions, whereas the internal differential methylation CpG sites (DMCs) of three types of
pluripotent cells tend to accumulate in the TSS1500 region. Furthermore, a series of endogenous
markers of cell reprogramming were identified based on the integrative analysis, including focal
adhesion, pluripotency maintenance and transcription regulation. The calcium signaling pathway
was detected as one of the signatures between NT cells and iPS cells. Finally, the regional bias of
DNA methylation for key pluripotency factors was discussed. Our studies provide new insight into
the barrier identification of cell reprogramming.
Conclusion:
Our studies analyzed some epigenetic markers and barriers of nuclear reprogramming,
hoping to provide new insight into understanding the underlying molecular mechanism
of reprogramming.
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Affiliation(s)
- Yongchun Zuo
- The College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
| | - Mingmin Song
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010070, China
| | - Hanshuang Li
- State key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010070, China
| | - Xing Chen
- State key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010070, China
| | - Pengbo Cao
- State key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010070, China
| | - Lei Zheng
- State key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010070, China
| | - Guifang Cao
- The College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
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49
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Klobučar T, Kreibich E, Krueger F, Arez M, Pólvora-Brandão D, von Meyenn F, da Rocha ST, Eckersley-Maslin M. IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted regions. Nucleic Acids Res 2020; 48:e92. [PMID: 32621604 PMCID: PMC7498334 DOI: 10.1093/nar/gkaa567] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2020] [Revised: 06/16/2020] [Accepted: 07/02/2020] [Indexed: 12/19/2022] Open
Abstract
Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
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Affiliation(s)
- Tajda Klobučar
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | - Elisa Kreibich
- Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK
| | - Felix Krueger
- Bioinformatics Group, Babraham Institute, Cambridge CB22 3AT, UK
| | - Maria Arez
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | - Duarte Pólvora-Brandão
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | | | - Simão Teixeira da Rocha
- Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
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50
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Huang CY, Li LH, Hsu WT, Cheng YC, Nicholson MW, Liu CL, Ting CY, Ko HW, Syu SH, Wen CH, Yan Z, Huang HP, Su HL, Chiang PM, Shen CN, Chen HF, Yen BLJ, Lu HE, Hwang SM, Chiou SH, Ho HN, Wu JY, Kamp TJ, Wu JC, Hsieh PCH. Copy number variant hotspots in Han Taiwanese population induced pluripotent stem cell lines - lessons from establishing the Taiwan human disease iPSC Consortium Bank. J Biomed Sci 2020; 27:92. [PMID: 32887585 PMCID: PMC7487458 DOI: 10.1186/s12929-020-00682-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Accepted: 08/24/2020] [Indexed: 11/15/2022] Open
Abstract
Background The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan’s growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. Methods We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. Results In the iPSCs, we identified ten specific CNV loci and seven “polymorphic” CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. Conclusions The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.
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Affiliation(s)
- Ching-Ying Huang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | - Ling-Hui Li
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | - Wan-Tseng Hsu
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
| | - Yu-Che Cheng
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | | | - Chun-Lin Liu
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | - Chien-Yu Ting
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | - Hui-Wen Ko
- Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, 300, Taiwan
| | - Shih-Han Syu
- Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, 300, Taiwan
| | - Cheng-Hao Wen
- Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, 300, Taiwan
| | - Zhuge Yan
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Hsiang-Po Huang
- Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
| | - Hong-Lin Su
- Department of Life Sciences, National Chung-Hsing University, Taichung, 402, Taiwan
| | - Po-Min Chiang
- Institute of Clinical Medicine, National Cheng Kung University, Tainan, 701, Taiwan
| | - Chia-Ning Shen
- Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan
| | - Hsin-Fu Chen
- Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
| | - B Lin Ju Yen
- Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, 350, Taiwan
| | - Huai-En Lu
- Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, 300, Taiwan
| | - Shiaw-Min Hwang
- Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, 300, Taiwan
| | - Shih-Hwa Chiou
- Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, 112, Taiwan
| | - Hong-Nerng Ho
- Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan
| | - Jer-Yuarn Wu
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
| | - Timothy J Kamp
- Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Joseph C Wu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Patrick C H Hsieh
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan.
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