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Zhang W, An S, Hou S, He X, Xiang J, Yan H, Liu X, Dong L, Wang X, Yang Y. Generation of transient totipotent blastomere-like stem cells by short-term high-dose Pladienolide B treatment. SCIENCE CHINA. LIFE SCIENCES 2025; 68:1337-1351. [PMID: 40024996 DOI: 10.1007/s11427-024-2774-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Accepted: 10/10/2024] [Indexed: 03/04/2025]
Abstract
As an alternative model for studying the dynamic process of early mammalian embryonic development, much progress has been made in using mouse embryonic stem cells (mESCs) to generate embryo-like structures, especially by modifying the starting cells. A previous study has demonstrated that totipotent blastomere-like cells (TBLCs) can be obtained by continuous treatment of mESCs with a low-dose splicing inhibitor, Pladienolide B (PlaB). However, these totipotent mESCs have limited proliferative capacity. Here, we report that short-term high-dose PlaB treatment can also induce mESCs to acquire totipotency. This treatment equips this novel type of stem cells with the ability to self-organize into blastoids and recapitulate key preimplantation developmental processes. Therefore, the stem cells are termed transient totipotent blastomere-like stem cells (tTBLCs). Transcriptome analysis showed that tTBLC blastoids bore similarities to mouse E3.5 blastocysts, E4.5 blastocysts, and TBLC blastoids. Additionally, we found that tTBLC blastoids could develop beyond the implantation stage, forming egg-cylinder-like structures both in vitro and in vivo. In summary, our research provides an alternative rapid and convenient method to generate the starting cells capable of developing into blastoids, which have immense application in various fields, not only in the basic study of early mouse embryogenesis but also in high-throughput drug screening.
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Affiliation(s)
- Wenyi Zhang
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Shiyu An
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Shuyue Hou
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Xingsi He
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Jinfeng Xiang
- Fourth Clinical Medicine College, Nanjing Medical University, Nanjing, 210004, China
- Department of Obstetrics, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Institute, Nanjing, 210004, China
| | - Huanyu Yan
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Xiaorui Liu
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Lingling Dong
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China
| | - Xi Wang
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China.
- Department of Prenatal Diagnosis of the First Affiliated Hospital of Nanjing Medical University, Nanjing, 210036, China.
| | - Yang Yang
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China.
- Innovation Center of Suzhou Nanjing Medical University, Suzhou, 215000, China.
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2
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Stapornwongkul KS, Hahn E, Poliński P, Salamó Palau L, Arató K, Yao L, Williamson K, Gritti N, Anlas K, Osuna Lopez M, Patil KR, Heemskerk I, Ebisuya M, Trivedi V. Glycolytic activity instructs germ layer proportions through regulation of Nodal and Wnt signaling. Cell Stem Cell 2025; 32:744-758.e7. [PMID: 40245870 PMCID: PMC12048219 DOI: 10.1016/j.stem.2025.03.011] [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: 12/20/2023] [Revised: 10/29/2024] [Accepted: 03/20/2025] [Indexed: 04/19/2025]
Abstract
Metabolic pathways can influence cell fate decisions, yet their regulative role during embryonic development remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in regulating signaling pathways involved in mesoderm and endoderm specification. Using a mouse embryonic stem cell (mESC)-based in vitro model for gastrulation, we found that glycolysis inhibition increases ectodermal cell fates at the expense of mesodermal and endodermal lineages. We demonstrate that this relationship is dose dependent, enabling metabolic control of germ layer proportions through exogenous glucose levels. We further show that glycolysis acts as an upstream regulator of Nodal and Wnt signaling and that its influence on cell fate specification can be decoupled from its effects on growth. Finally, we confirm the generality of our findings using a human gastrulation model. Our work underscores the dependence of signaling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision-making.
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Affiliation(s)
- Kristina S Stapornwongkul
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain.
| | - Elisa Hahn
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | - Patryk Poliński
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | - Laura Salamó Palau
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | - Krisztina Arató
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | - LiAng Yao
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Kate Williamson
- Medical Research Council Toxicology Unit, University of Cambridge, Cambridge CB2 1QR, UK
| | - Nicola Gritti
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | - Kerim Anlas
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain
| | | | - Kiran R Patil
- Medical Research Council Toxicology Unit, University of Cambridge, Cambridge CB2 1QR, UK
| | - Idse Heemskerk
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Physics, University of Michigan, Ann Arbor, MI 48109, USA
| | - Miki Ebisuya
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain; Cluster of Excellence Physics of Life, TU Dresden, 01307 Dresden, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
| | - Vikas Trivedi
- European Molecular Biology Laboratory, EMBL Barcelona, C/ Dr. Aiguader, 88, PRBB Building, 08003 Barcelona, Spain; Developmental Biology, EMBL Heidelberg, Heidelberg 69117, Germany.
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3
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Lee EJ, Sun R, Kim J. The self-renewal function of Oct-4 can be replaced by the EWS-Oct-4 fusion protein in embryonic stem cells. Cell Mol Life Sci 2025; 82:166. [PMID: 40251420 PMCID: PMC12008092 DOI: 10.1007/s00018-025-05701-0] [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: 11/26/2024] [Revised: 03/31/2025] [Accepted: 04/07/2025] [Indexed: 04/20/2025]
Abstract
Octamer-binding transcription factor 4 (Oct-4) is essential for maintenance and pluripotency of embryonic stem (ES) cells. Despite the structural similarities between Oct-4 and its homologs (Oct-1, Oct-2, and Oct-6), these homologs cannot serve as substitutes for Oct-4 when generating stem cell colonies. While nuclear receptor subfamily 5, group A, member 2 (Nr5a2) can temporarily serve as a substitute for Oct-4 during cellular reprogramming, it is insufficient to maintain these functions in ES cells. The EWS-Oct-4 fusion protein, which was identified in human tumors, is a viable alternative that can potentially sustain and enhance ES cell functions. This study used ZHBTc4 ES cells, which have tetracycline-regulated Oct-4 expression, to explore the capabilities of EWS-Oct-4. It employed a variety of assays, including western blotting, immunocytochemistry, RT-PCR, luciferase reporter assays, flow cytometry, and teratoma formation assays. EWS-Oct-4 preserved the self-renewal capacity of Oct-4-null ES cells, as demonstrated by their undifferentiated morphology and increased expression of pluripotency markers such as Sox2, Nanog, and SSEA-1. It also boosted cell proliferation and influenced cell cycle dynamics by downregulating p21 and upregulating Oct-4 target genes, including Rex-1 and fibroblast growth factor-4. Epithelial markers were upregulated and mesenchymal markers were downregulated, suggesting a shift toward an epithelial phenotype. Prominent teratoma formation further confirmed the functionality of EWS-Oct-4 in vivo. The integrity and specific functional domains of EWS-Oct-4 were critical for these effects. Finally, comparative transcriptomic analysis revealed that ES cells expressing EWS-Oct-4 and those expressing Oct-4 had highly similar global gene expression profiles, with distinct variations in differentially expressed genes. These findings indicate that EWS-Oct-4 can effectively replace Oct-4, which has significant implications for advancements in stem cell research and regenerative medicine.
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Affiliation(s)
- Eun Joo Lee
- Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, 04107, Korea
| | - Ruijing Sun
- Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, 04107, Korea
| | - Jungho Kim
- Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, 04107, Korea.
- Stress-Responding Bionanomaterial Center, Sogang University, Seoul, 04107, Korea.
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4
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Cuomo M, Costabile D, Della Monica R, Buonaiuto M, Trio F, De Riso G, Visconti R, Chiariotti L. A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer-promoter interaction. Nucleic Acids Res 2025; 53:gkaf274. [PMID: 40219964 PMCID: PMC11992674 DOI: 10.1093/nar/gkaf274] [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: 07/19/2024] [Revised: 03/20/2025] [Accepted: 03/25/2025] [Indexed: 04/14/2025] Open
Abstract
Despite a plethora of studies exploring the transcriptional regulation of the Nanog gene, the role of the enhancer RNAs (eRNAs) derived from Nanog-interacting super-enhancers (SEs) remains under-investigated. In the present study, we examined the functional role of the eRNAs transcribed from the -5 kb Nanog SE in mouse embryonic stem cells (mESCs) and found that an eRNA, here defined as -5KNAR, was essential to maintain the Nanog locus in an epigenetically active configuration, thereby ensuring pluripotency. We found that the here identified -5KNAR functionally interacts with the RAD21 protein, suggesting a role in stabilizing a cohesin complex at the Nanog locus, ensuring the generation and maintenance of an enhancer-promoter loop. Silencing of -5KNAR caused a cascade of events, including the generation of a DNA methylation wave (likely spreading from a single methylated CpG site), substantial chromatin remodeling, and loss of the enhancer-promoter loop, inducing Nanog silencing and mESC differentiation. Under these conditions, exogenous re-expression of Nanog was unable to restore either the endogenous Nanog expression or the enhancer-promoter interaction, suggesting that, at hierarchical level, the expression of the -5KNAR plays a prominent role in maintaining the pluripotency in mESCs.
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Affiliation(s)
- Mariella Cuomo
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Via S. Pansini 5, Naples 80131, Italy
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
| | - Davide Costabile
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
- SEMM-European School of Molecular Medicine, Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Naples 80131, Italy
| | - Rosa Della Monica
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Via S. Pansini 5, Naples 80131, Italy
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
| | - Michela Buonaiuto
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Via S. Pansini 5, Naples 80131, Italy
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
| | - Federica Trio
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
| | - Giulia De Riso
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Via S. Pansini 5, Naples 80131, Italy
| | - Roberta Visconti
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
- Institute for the Experimental Endocrinology and Oncology “G. Salvatore”, Italian National Council of Research, Via S. Pansini 5, 80131 Naples, Italy
| | - Lorenzo Chiariotti
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Via S. Pansini 5, Naples 80131, Italy
- CEINGE Advanced Biotechnologies “Franco Salvatore”, Naples 80145, Italy
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5
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Li M, Jiang Z, Xu X, Wu X, Liu Y, Chen K, Liao Y, Li W, Wang X, Guo Y, Zhang B, Wen L, Kee K, Tang F. Chromatin accessibility landscape of mouse early embryos revealed by single-cell NanoATAC-seq2. Science 2025; 387:eadp4319. [PMID: 40146829 DOI: 10.1126/science.adp4319] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Accepted: 01/13/2025] [Indexed: 03/29/2025]
Abstract
In mammals, fertilized eggs undergo genome-wide epigenetic reprogramming to generate the organism. However, our understanding of epigenetic dynamics during preimplantation development at single-cell resolution remains incomplete. Here, we developed scNanoATAC-seq2, a single-cell assay for transposase-accessible chromatin using long-read sequencing for scarce samples. We present a detailed chromatin accessibility landscape of mouse preimplantation development, revealing distinct chromatin signatures in the epiblast, primitive endoderm, and trophectoderm during lineage segregation. Differences between zygotes and two-cell embryos highlight reprogramming in chromatin accessibility during the maternal-to-zygotic transition. Single-cell long-read sequencing enables in-depth analysis of chromatin accessibility in noncanonical imprinting, imprinted X chromosome inactivation, and low-mappability genomic regions, such as repetitive elements and paralogs. Our data provide insights into chromatin dynamics during mammalian preimplantation development and lineage differentiation.
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Affiliation(s)
- Mengyao Li
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
- The State Key Laboratory for Complex, Severe, and Rare Diseases; School of Basic Medical Sciences, Tsinghua Medicine, Tsinghua University, Beijing, China
| | - Zhenhuan Jiang
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Xueqiang Xu
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Xinglong Wu
- College of Animal Science and Technology, Hebei Agricultural University, Baoding, Hebei , China
| | - Yun Liu
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- Changping Laboratory, Beijing, China
| | - Kexuan Chen
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Yuhan Liao
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Wen Li
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- Changping Laboratory, Beijing, China
| | - Xiao Wang
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Yuqing Guo
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Bo Zhang
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Lu Wen
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
| | - Kehkooi Kee
- PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
- The State Key Laboratory for Complex, Severe, and Rare Diseases; School of Basic Medical Sciences, Tsinghua Medicine, Tsinghua University, Beijing, China
- SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Shanxi Medical University, Taiyuan, Shanxi Province, China
| | - Fuchou Tang
- Biomedical Pioneering Innovative Center, School of Life Sciences, Peking University, Beijing, China
- New Cornerstone Science Laboratory, Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- Changping Laboratory, Beijing, China
- PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
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6
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Battaglia R, Caponnetto A, Ferrara C, Fazzio A, Barbagallo C, Stella M, Barbagallo D, Ragusa M, Vento ME, Borzì P, Scollo P, Carli L, Feichtinger M, Kasapi E, Tsakos E, Palini S, Sierka W, Pecorino B, Campitiello MR, Ronsini C, Purrello M, Valerio D, Longobardi S, D'Hooghe T, Di Pietro C. Up-regulated microRNAs in blastocoel fluid of human implanted embryos could control circuits of pluripotency and be related to embryo competence. J Assist Reprod Genet 2025:10.1007/s10815-025-03457-x. [PMID: 40140178 DOI: 10.1007/s10815-025-03457-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Accepted: 03/14/2025] [Indexed: 03/28/2025] Open
Abstract
PURPOSE The paper aims to investigate the biological role of microRNAs secreted by preimplantation embryo into the blastocoel fluid and to detect a distinctive molecular signature for identifying embryos with the highest implantation potential. METHODS We carried on a multicenter retrospective study involving five European IVF centers. We collected 112 blastocoel fluid samples from embryos on day 5 post-fertilization, cultured individually, along with data on blastocyst grade and embryo transfer outcomes. Using a custom TLDA Array, we compared the expression levels of 89 miRNAs between 33 fluids from high-quality implanted embryos and 30 fluids from high-quality not-implanted embryos. Expression differences were assessed using SAM and t-test. Additionally, correlation and function enrichment analysis and network construction were conducted to identify the biological roles of deregulated microRNAs. RESULTS We identified six up-regulated microRNAs in the blastocoel fluid from implanted embryos, significantly and positively correlated across all samples (r ≥ 0.7; P ≤ 0.05). They could take part in pluripotency circuits, regulating and being regulated by transcription factors associated with stemness, cell growth, and embryo development. The ROC curve analysis confirmed the potential of these miRNAs as implantation classifiers. CONCLUSION The six miRNAs up-regulated in blastocoel fluid from implanted embryos may represent a functional molecular signature for evaluating blastocyst quality and identifying the most competent embryos. Their evaluation associated with non-invasive preimplantation genetic testing, integrating epigenetic and genomic analyses, could enhance implantation grade and allow for identification of the euploid embryo not able to implant.
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Affiliation(s)
- Rosalia Battaglia
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
- Department of Medicine and Surgery, University of Enna "Kore", 94100, Enna, Italy
| | - Angela Caponnetto
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Carmen Ferrara
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Anna Fazzio
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Cristina Barbagallo
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Michele Stella
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Davide Barbagallo
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | - Marco Ragusa
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | | | | | - Paolo Scollo
- Department of Medicine and Surgery, University of Enna "Kore", 94100, Enna, Italy
- IVF Unit, Cannizzaro Hospital, Catania, Italy
| | - Luca Carli
- Wunschbaby Institut Feichtinger, Vienna, Austria
| | | | | | - Elias Tsakos
- EmbryoClinic IVF, Kalamaria, Thessaloniki, Greece
| | | | | | - Basilio Pecorino
- Department of Medicine and Surgery, University of Enna "Kore", 94100, Enna, Italy
| | - Maria Rosaria Campitiello
- Department of Obstetrics and Gynecology and Physiopathology of Human Reproduction, ASL Salerno, Salerno, Italy
| | - Carlo Ronsini
- Department of Woman, Child and General and Specialized Surgery, University of Campania "Luigi Vanvitelli", Naples, Italy
| | - Michele Purrello
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy
| | | | | | - Thomas D'Hooghe
- Merck KGaA, Darmstadt, Germany
- Department of Development and Regeneration, Biomedical Sciences Group, KU Leuven (University of Leuven), Leuven, Belgium
| | - Cinzia Di Pietro
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123, Catania, Italy.
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7
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Guo Z, Yao J, Zheng X, Cao J, Lv X, Gao Z, Guo S, Li H, Guan D, Li L, Qin D, Li D, Wang X, Tan M, Zhang J, Zhang Y, Wang B, Bu W, Li J, Zhao X, Meng F, Feng Y, Li L, Du J, Fan Y. Cavity oscillation drives pattern formation in early mammalian embryos. Cell Rep 2025; 44:115342. [PMID: 39985766 DOI: 10.1016/j.celrep.2025.115342] [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: 04/23/2024] [Revised: 10/02/2024] [Accepted: 01/31/2025] [Indexed: 02/24/2025] Open
Abstract
During the second cell fate in mouse embryos, the inner cell mass (ICM) segregates into the spatially distinct epiblast (EPI) and primitive endoderm (PrE) layers. The mechanism driving this pattern formation, however, remains unresolved. Here, we report that, concomitant with the segregation process of EPI/PrE precursors starting from mid-blastocyst, the blastocyst cavity begins to oscillate cyclically with rapid contraction yet slow expansion, triggering a phase transition in the ICM to a fluid-like state. This asymmetric oscillation of the blastocyst cavity facilitates EPI/PrE segregation by enhancing cell-cell contact fluctuations within the ICM and initiating convergent cell flows, which induce movement of these two cell types in opposite directions, wherein PrE precursors move toward the ICM-lumen interface, whereas EPI precursors move toward the trophectoderm. Last, we found that both PDGFRα expression and YAP nuclear accumulation in PrE precursors increase in response to blastocyst cavity oscillation. This study reveals the foundational role of physical oscillation in driving embryonic pattern formation during early mammalian embryonic development.
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Affiliation(s)
- Zheng Guo
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Jie Yao
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Xu Zheng
- State Key Laboratory of Nonlinear Mechanics, Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China
| | - Jialing Cao
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Xinxin Lv
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Zheng Gao
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China
| | - Shuyu Guo
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Hangyu Li
- State Key Laboratory of Nonlinear Mechanics, Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China
| | - Dongshi Guan
- State Key Laboratory of Nonlinear Mechanics, Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China
| | - Long Li
- State Key Laboratory of Nonlinear Mechanics, Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China
| | - Dandan Qin
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Dong Li
- Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
| | - Xiaoxiao Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Min Tan
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Jing Zhang
- Laboratory Animal Research Center, Tsinghua University, Beijing 100084, China
| | - Yanli Zhang
- Imaging Core Facility, Technology Center for Protein Science, Tsinghua University, Beijing 100084, China
| | - Bo Wang
- Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xi'ning 810008, China
| | - Wanjuan Bu
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Jianwen Li
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Xinbin Zhao
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Fanzhe Meng
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Yue Feng
- Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
| | - Lei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jing Du
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China.
| | - Yubo Fan
- Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China.
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8
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Crowley D, Simpson L, Chatfield J, Forey T, Allegrucci C, Sang F, Holmes N, Genikhovich G, Technau U, Cunningham D, Silva E, Mullin N, Dixon JE, Loose M, Alberio R, Johnson AD. Programming of pluripotency and the germ line co-evolved from a Nanog ancestor. Cell Rep 2025; 44:115396. [PMID: 40057954 DOI: 10.1016/j.celrep.2025.115396] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Revised: 12/21/2024] [Accepted: 02/13/2025] [Indexed: 03/29/2025] Open
Abstract
Francois Jacob proposed that evolutionary novelty arises through incremental tinkering with pre-existing genetic mechanisms. Vertebrate evolution was predicated on pluripotency, the ability of embryonic cells to form somatic germ layers and primordial germ cells (PGCs). The origins of pluripotency remain unclear, as key regulators, such as Nanog, are not conserved outside of vertebrates. Given NANOG's role in mammalian development, we hypothesized that NANOG activity might exist in ancestral invertebrate genes. Here, we find that Vent from the hemichordate Saccoglossus kowalevskii exhibits NANOG activity, programming pluripotency in Nanog-/- mouse pre-induced pluripotent stem cells (iPSCs) and NANOG-depleted axolotl embryos. Vent from the cnidarian Nematostella vectensis showed partial activity, whereas Vent from sponges and vertebrates had no activity. VENTX knockdown in axolotls revealed a role in germline-competent mesoderm, which Saccoglossus Vent could rescue but Nematostella Vent could not. This suggests that the last deuterostome ancestor had a Vent gene capable of programming pluripotency and germline competence.
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Affiliation(s)
- Darren Crowley
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
| | - Luke Simpson
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Jodie Chatfield
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Teri Forey
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Cinzia Allegrucci
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Fei Sang
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Nadine Holmes
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Grigory Genikhovich
- Department of Neurosciences and Developmental Biology, Faculty of Life Sciences, Vienna BioCenter, Djerassiplatz 1, 1030 Vienna, Austria
| | - Ulrich Technau
- Department of Neurosciences and Developmental Biology, Faculty of Life Sciences, Vienna BioCenter, Djerassiplatz 1, 1030 Vienna, Austria
| | | | - Elena Silva
- Department of Biology, Georgetown University, Washington, D.C, USA
| | - Nicholas Mullin
- Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, UK
| | - James E Dixon
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Matthew Loose
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
| | - Andrew D Johnson
- School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK
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9
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Kavaklioglu G, Podhornik A, Vcelkova T, Marjanovic J, Beck MA, Phan-Canh T, Mair T, Miccolo C, Drino A, Doni M, Egger G, Chiocca S, Modic M, Seiser C. The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition. eLife 2025; 13:RP96850. [PMID: 40112032 PMCID: PMC11925450 DOI: 10.7554/elife.96850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/22/2025] Open
Abstract
Repression of retrotransposition is crucial for the successful fitness of a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, is an RNA-binding protein that is expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it is found to be essential for maintaining pluripotency. In cancer, L1TD1 expression is highly correlative with malignancy progression and as such considered a potential prognostic factor for tumors. However, its molecular role in cancer remains largely unknown. Our findings reveal that DNA hypomethylation induces the expression of L1TD1 in HAP1 human tumor cells. L1TD1 depletion significantly modulates both the proteome and transcriptome and thereby reduces cell viability. Notably, L1TD1 associates with L1 transcripts and interacts with L1 ORF1p protein, thereby facilitating L1 retrotransposition. Our data suggest that L1TD1 collaborates with its ancestral L1 ORF1p as an RNA chaperone, ensuring the efficient retrotransposition of L1 retrotransposons, rather than directly impacting the abundance of L1TD1 targets. In this way, L1TD1 might have an important role not only during early development but also in tumorigenesis.
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Affiliation(s)
- Gülnihal Kavaklioglu
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Alexandra Podhornik
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Terezia Vcelkova
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Jelena Marjanovic
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Mirjam A Beck
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Trinh Phan-Canh
- Medical University of Vienna, Max Perutz Labs Vienna, Campus Vienna BiocenterViennaAustria
| | - Theresia Mair
- Department of Pathology, Medical University of ViennaViennaAustria
| | - Claudia Miccolo
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCSMilanItaly
| | - Aleksej Drino
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
| | - Mirko Doni
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCSMilanItaly
| | - Gerda Egger
- Department of Pathology, Medical University of ViennaViennaAustria
| | - Susanna Chiocca
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCSMilanItaly
| | - Miha Modic
- National Institute of ChemistryLjubljanaSlovenia
- Dementia Research Institute at King’s College London and The Francis Crick instituteLondonUnited Kingdom
| | - Christian Seiser
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of ViennaViennaAustria
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10
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Allouh MZ, Rizvi SFA, Alamri A, Jimoh Y, Aouda S, Ouda ZH, Hamad MIK, Perez-Cruet M, Chaudhry GR. Mesenchymal stromal/stem cells from perinatal sources: biological facts, molecular biomarkers, and therapeutic promises. Stem Cell Res Ther 2025; 16:127. [PMID: 40055783 PMCID: PMC11889844 DOI: 10.1186/s13287-025-04254-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 02/25/2025] [Indexed: 05/13/2025] Open
Abstract
The use of mesenchymal stem cells (MSCs) from perinatal tissue sources has gained attention due to their availability and lack of significant ethical or moral concerns. These cells have a higher proliferative capability than adult MSCs and less immunogenic or tumorigenesis risk than fetal and embryonic stem cells. Additionally, they do not require invasive isolation methods like fetal and adult MSCs. We reviewed the main biological and therapeutic aspects of perinatal MSCs in a three-part article. In the first part, we revised the main biological features and characteristics of MSCs and the advantages of perinatal MSCs over other types of SCs. In the second part, we provided a detailed molecular background for the main biomarkers that can be used to identify MSCs. In the final part, we appraised the therapeutic application of perinatal MSCs in four major degenerative disorders: degenerative disc disease, retinal degenerative diseases, ischemic heart disease, and neurodegenerative diseases. In conclusion, there is no single specific molecular marker to identify MSCs. We recommend using at least two positive markers of stemness (CD29, CD73, CD90, or CD105) and two negative markers (CD34, CD45, or CD14) to exclude the hematopoietic origin. Moreover, utilizing perinatal MSCs for managing degenerative diseases presents a promising therapeutic approach. This review emphasizes the significance of employing more specialized progenitor cells that originated from the perinatal MSCs. The review provides scientific evidence from the literature that applying these progenitor cells in therapeutic procedures provides a greater regenerative capacity than the original primitive MSCs. Finally, this review provides a valuable reference for researchers exploring perinatal MSCs and their therapeutic applications.
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Affiliation(s)
- Mohammed Z Allouh
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE.
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA.
| | - Syed Faizan Ali Rizvi
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Ali Alamri
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Yusuf Jimoh
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Salma Aouda
- College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, UAE
| | - Zakaria H Ouda
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE
| | - Mohammad I K Hamad
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE
| | - Mick Perez-Cruet
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Neurosurgery, Corewell Health, Royal Oak, MI, USA
| | - G Rasul Chaudhry
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA.
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA.
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11
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Kim N, Yokobayashi Y. Scalable control of stem cell fate by riboswitch-regulated RNA viral vector without genomic integration. Mol Ther 2025; 33:1213-1225. [PMID: 39797398 PMCID: PMC11897756 DOI: 10.1016/j.ymthe.2025.01.005] [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: 02/01/2024] [Revised: 07/30/2024] [Accepted: 01/08/2025] [Indexed: 01/13/2025] Open
Abstract
Transgene expression in stem cells is a powerful means of regulating cellular properties and differentiation into various cell types. However, existing vectors for transgene expression in stem cells suffer from limitations such as the need for genomic integration, the transient nature of gene expression, and the inability to temporally regulate transgene expression, which hinder biomedical and clinical applications. Here we report a new class of RNA virus-based vectors for scalable and integration-free transgene expression in mouse embryonic stem cells (mESCs). The vector is equipped with a small molecule-regulated riboswitch and a drug selection marker that allow temporal regulation of transgene expression and stable maintenance of the vector in proliferating stem cells. We demonstrated the utility of the vector by maintaining the pluripotency of mESCs in a differentiation induction medium by expressing Nanog and inducing myogenic differentiation by triggering Myod1 expression, without altering the mESC genome.
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Affiliation(s)
- Narae Kim
- Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan
| | - Yohei Yokobayashi
- Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan.
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12
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Athanasouli P, Vanhessche T, Lluis F. Divergent destinies: insights into the molecular mechanisms underlying EPI and PE fate determination. Life Sci Alliance 2025; 8:e202403091. [PMID: 39779220 PMCID: PMC11711469 DOI: 10.26508/lsa.202403091] [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: 10/14/2024] [Revised: 12/21/2024] [Accepted: 12/23/2024] [Indexed: 01/11/2025] Open
Abstract
Mammalian pre-implantation development is entirely devoted to the specification of extra-embryonic lineages, which are fundamental for embryo morphogenesis and support. The second fate decision is taken just before implantation, as defined by the epiblast (EPI) and the primitive endoderm (PE) specification. Later, EPI forms the embryo proper and PE contributes to the formation of the yolk sac. The formation of EPI and PE as molecularly and morphologically distinct lineages is the final step of a multistage process, which begins when bipotent progenitor cells diverge into separate fates. Despite advances in uncovering the molecular mechanisms underlying the differential transcriptional patterns that dictate how apparently identical cells make fate decisions and how lineage integrity is maintained, a detailed overview of these mechanisms is still lacking. In this review, we dissect the EPI and PE formation process into four stages (initiation, specification, segregation, and maintenance) and we provide a comprehensive understanding of the molecular mechanisms involved in lineage establishment in the mouse. In addition, we discuss the conservation of key processes in humans, based on the most recent findings.
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Affiliation(s)
- Paraskevi Athanasouli
- Department of Development and Regeneration, Stem Cell Institute, KU Leuven, Leuven, Belgium
| | - Tijs Vanhessche
- Department of Development and Regeneration, Stem Cell Institute, KU Leuven, Leuven, Belgium
| | - Frederic Lluis
- Department of Development and Regeneration, Stem Cell Institute, KU Leuven, Leuven, Belgium
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13
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Yuan F, Yang J, Ma F, Hu Z, Malik V, Zang R, Li D, Shi X, Huang X, Zhou H, Wang J. Pluripotency factor Tex10 finetunes Wnt signaling for spermatogenesis and primordial germ cell development. Nat Commun 2025; 16:1900. [PMID: 39988597 PMCID: PMC11847947 DOI: 10.1038/s41467-025-57165-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Accepted: 02/13/2025] [Indexed: 02/25/2025] Open
Abstract
Testis-specific transcript 10 (Tex10) is highly expressed in the testis, embryonic stem cells (ESCs), and primordial germ cells (PGCs). We previously generated a Tex10 knockout mouse model demonstrating its critical roles in ESC pluripotency and preimplantation development. Here, using conditional knockout mice and dTAG-degron ESCs, we show Tex10 is required for spermatogenesis and ESC-to-PGCLC differentiation. Specifically, Tex10-null spermatocytes arrest at metaphase I, compromising round spermatid formation. Tex10 depletion and overexpression compromise and enhance ESC-to-PGCLC differentiation, respectively. Mechanistically, bulk and single-cell RNA sequencing reveals that Tex10 depletion downregulates genes involved in pluripotency, PGC development, and spermatogenesis while upregulating genes promoting somatic programs. Chromatin occupancy study reveals that Tex10 binds to H3K4me3-marked promoters of Psmd3 and Psmd7, negative regulators of Wnt signaling, and activates their expression, thereby restraining Wnt signaling. Our study identifies Tex10 as a previously unappreciated factor in spermatogenesis and PGC development, offering potential therapeutic insights for treating male infertility.
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Affiliation(s)
- Feifei Yuan
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Jihong Yang
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
- BoYu Intelligent Health Innovation Laboratory, Hangzhou, China
| | - Fanglin Ma
- Department of Cell, Developmental and Regenerative Biology; The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Zhe Hu
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Vikas Malik
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Ruge Zang
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Dan Li
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Xianle Shi
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Xin Huang
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Hongwei Zhou
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Jianlong Wang
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia Stem Cell Initiative, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA.
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14
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Nielsen MI, Wolters JC, Bringas OGR, Jiang H, Di Stefano LH, Oghbaie M, Hozeifi S, Nitert MJ, van Pijkeren A, Smit M, Ter Morsche L, Mourtzinos A, Deshpande V, Taylor MS, Chait BT, LaCava J. Targeted detection of endogenous LINE-1 proteins and ORF2p interactions. Mob DNA 2025; 16:3. [PMID: 39915890 PMCID: PMC11800616 DOI: 10.1186/s13100-024-00339-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Accepted: 12/24/2024] [Indexed: 02/09/2025] Open
Abstract
BACKGROUND Both the expression and activities of LINE-1 (L1) retrotransposons are known to occur in numerous cell-types and are implicated in pathobiological contexts such as aging-related inflammation, autoimmunity, and in cancers. L1s encode two proteins that are translated from bicistronic transcripts. The translation product of ORF1 (ORF1p) has been robustly detected by immunoassays and shotgun mass spectrometry (MS). Yet, more sensitive detection methods would enhance the use of ORF1p as a clinical biomarker. In contrast, until now, no direct evidence of endogenous L1 ORF2 translation to protein (ORF2p) has been shown. Instead, assays for ORF2p have been limited to ectopic L1 ORF over-expression contexts and to indirect detection of endogenous ORF2p enzymatic activity, such as by the sequencing of de novo genomic insertions. Immunoassays for endogenous ORF2p have been problematic, producing apparent false positives due to cross-reactivities, and shotgun MS has not yielded reliable evidence of ORF2p peptides in biological samples. RESULTS Here we present targeted mass spectrometry assays, selected and parallel reaction monitoring (SRM and PRM, respectively) to detect and quantify L1 ORF1p and ORF2p at their endogenous abundances. We were able to quantify ORF1p and ORF2p present in our samples down to a range in the low attomoles. Confident in our ability to affinity enrich ORF2p, we describe an interactome associated with endogenous ORF2-containing macromolecular assemblies. CONCLUSIONS This is the first assay to demonstrate sensitive and robust quantitation of endogenous ORF2p. The ability to assay ORF2p directly and quantitatively will improve our understanding of the developmental and diseased cell states where L1 expression and its activity naturally occur. The ability to simultaneously assay endogenous L1 ORF1p and ORF2p is an important step forward for L1 analytical biochemistry. Endogenous ORF2p interactomes can now be presented with confidence that ORF2p is among the enriched proteins.
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Affiliation(s)
- Mathias I Nielsen
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
| | - Justina C Wolters
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
| | - Omar G Rosas Bringas
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Hua Jiang
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
| | - Luciano H Di Stefano
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Mehrnoosh Oghbaie
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Samira Hozeifi
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Mats J Nitert
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Alienke van Pijkeren
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Marieke Smit
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Lars Ter Morsche
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Apostolos Mourtzinos
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Vikram Deshpande
- Department of Pathology, Mass General Brigham and Harvard Medical School, Boston, MA, USA
| | - Martin S Taylor
- Department of Pathology, Mass General Brigham and Harvard Medical School, Boston, MA, USA
| | - Brian T Chait
- Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA
| | - John LaCava
- Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA.
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
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15
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Amissah HA, Antwi MH, Amissah TA, Combs SE, Shevtsov M. More than Just Protein Folding: The Epichaperome, Mastermind of the Cancer Cell. Cells 2025; 14:204. [PMID: 39936995 PMCID: PMC11817126 DOI: 10.3390/cells14030204] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2025] [Revised: 01/26/2025] [Accepted: 01/28/2025] [Indexed: 02/13/2025] Open
Abstract
The epichaperome, a dynamic and integrated network of chaperone proteins, extends its roles beyond basic protein folding to protein stabilization and intracellular signal transduction to orchestrating a multitude of cellular processes critical for tumor survival. In this review, we explore the multifaceted roles of the epichaperome, delving into its diverse cellular locations, factors that modulate its formation and function, its liquid-liquid phase separation, and the key signaling and crosstalk pathways it regulates, including cellular metabolism and intracellular signal transduction. We further highlight techniques for isolating and identifying epichaperome networks, pitfalls, and opportunities. Further, we review the profound implications of the epichaperome for cancer treatment and therapy design, underscoring the need for strategic engineering that hinges on a comprehensive insight into the comprehensive structure and workings of the epichaperome across the heterogeneous cell subpopulations in the tumor milieu. By presenting a holistic view of the epichaperome's functions and mechanisms, we aim to underscore its potential as a key target for novel anti-cancer strategies, revealing that the epichaperome is not merely a piece of protein folding machinery but a mastermind that facilitates the malignant phenotype.
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Affiliation(s)
- Haneef Ahmed Amissah
- Institute of Life Sciences and Biomedicine, Department of Medical Biology and Biotechnology, School of Medicine and Life Sciences, Far Eastern Federal University, Vladivostok 690922, Russia;
- Diagnostics Laboratory Department, Trauma and Specialist Hospital, Winneba CE-122-2486, Central Region, Ghana
| | - Maxwell Hubert Antwi
- Department of Medical Laboratory Science, Faculty of Health and Allied Sciences, Koforidua Technical University, Koforidua EN-112-3991, Eastern Region, Ghana; (M.H.A.); (T.A.A.)
| | - Tawfeek Ahmed Amissah
- Department of Medical Laboratory Science, Faculty of Health and Allied Sciences, Koforidua Technical University, Koforidua EN-112-3991, Eastern Region, Ghana; (M.H.A.); (T.A.A.)
| | - Stephanie E. Combs
- Department of Radiation Oncology, Technische Universität München (TUM), Klinikum Rechts der Isar, 81675 Munich, Germany;
| | - Maxim Shevtsov
- Department of Radiation Oncology, Technische Universität München (TUM), Klinikum Rechts der Isar, 81675 Munich, Germany;
- Laboratory of Biomedical Nanotechnologies, Institute of Cytology of the Russian Academy of Sciences (RAS), Saint Petersburg 194064, Russia
- Personalized Medicine Centre, Almazov National Medical Research Centre, Saint Petersburg 197341, Russia
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16
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Radoszkiewicz K, Rybkowska P, Szymanska M, Krzesniak NE, Sarnowska A. The influence of biomimetic conditions on neurogenic and neuroprotective properties of dedifferentiated fat cells. Stem Cells 2025; 43:sxae066. [PMID: 39576128 PMCID: PMC11811640 DOI: 10.1093/stmcls/sxae066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2024] [Accepted: 09/30/2024] [Indexed: 02/12/2025]
Abstract
In the era of a constantly growing number of reports on the therapeutic properties of dedifferentiated, ontogenetically rejuvenated cells and their use in the treatment of neurological diseases, the optimization of their derivation and long-term culture methods seem to be crucial. One of the solutions is seen in the use of dedifferentiated fat cells (DFATs) that are characterized by a greater homogeneity. Moreover, these cells seem to possess a higher expression of transcriptional factors necessary to maintain pluripotency (stemness-related transcriptional factors) as well as a greater ability to differentiate in vitro into 3 embryonic germ layers, and a high proliferative potential in comparison to adipose stem/stromal cells. However, the neurogenic and neuroprotective potential of DFATs is still insufficiently understood; hence, our research goal was to contribute to our current knowledge of the subject. To recreate the brain's physiological (biomimetic) conditions, the cells were cultured at 5% oxygen concentration. The neural differentiation capacity of DFATs was assessed in the presence of the N21 supplement containing the factors that are typically found in the natural environment of the neural cell niche or in the presence of cerebrospinal fluid and under various spatial conditions (microprinting). The neuroprotective properties of DFATs were assessed using the coculture method with the ischemically damaged nerve tissue.
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Affiliation(s)
- Klaudia Radoszkiewicz
- Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02‐106 Warsaw, Poland
| | - Paulina Rybkowska
- Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02‐106 Warsaw, Poland
| | - Magdalena Szymanska
- Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02‐106 Warsaw, Poland
| | - Natalia Ewa Krzesniak
- Department of Plastic and Reconstructive Surgery, Centre of Postgraduate Medical Education, Prof. W. Orlowski Memorial Hospital, 00‐416 Warsaw, Poland
| | - Anna Sarnowska
- Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02‐106 Warsaw, Poland
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17
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Hassan S, Ashraf N, Hanif K, Khan NU. Subcortical Maternal Complex in Female Infertility: A Transition from Animal Models to Human Studies. Mol Biol Rep 2025; 52:108. [PMID: 39775990 DOI: 10.1007/s11033-025-10220-z] [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: 10/15/2024] [Accepted: 01/02/2025] [Indexed: 01/11/2025]
Abstract
Female infertility is a significant healthcare burden that is frequently encountered among couples globally. While environmental factors, comorbidities, and lifestyle determine reproductive health, certain genetic variants in key reproductive genes can potentially cause unsuccessful pregnancies. Such crucial proteins have been identified within the subcortical maternal complex (SCMC) and play an integral role in the early stages of embryogenesis before embryo implantation. SCMC proteins are associated with crucial pathways during embryogenesis, causing changes that are necessary for the transition of an oocyte to an embryo. These vital processes include the formation of cytoplasmic spindles and lattices, accurate positioning of meiotic spindles, regulatory roles in various gene translations, organelle redistribution, and zygotic genome reprogramming. While these genes are well studied in animal models, often mice, translation to clinical studies is comparatively less. The present study elucidates the transition in genetic studies from animal to human models of SCMC proteins. The present literature review shows that the expression of various SCMC proteins impairs embryo development at different stages. The clinical translation of SCMC occurs via various pathways. Therefore, females experiencing multiple unsuccessful pregnancies after natural or assisted conception techniques are candidates for underlying SCMC mutations. Although the phenotype of affected individuals has been identified, the molecular mechanisms that lead to impaired pathways still require investigation. Therefore, the present study paves the way for future research leading to the early diagnosis of lethal variants and possible subsequent management.
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Affiliation(s)
- Sibte Hassan
- Reproductive Medicine Physician SEHA Corniche Hospital, Abu Dhabi, UAE.
| | - Nomia Ashraf
- Department of obstetrics and gynaecology, Fatima Jinnah Medical University Lahore, Lahore, Pakistan
| | - Khola Hanif
- Genova Invitro Fertilization Clinic Lahore, Lahore, Pakistan
| | - Najeeb Ullah Khan
- Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Pakistan.
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18
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Hirono N, Hashimoto M, Shimojo H, Sasaki H. Fate specification triggers a positive feedback loop of TEAD-YAP and NANOG to promote epiblast formation in preimplantation embryos. Development 2025; 152:dev203091. [PMID: 39629521 DOI: 10.1242/dev.203091] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2024] [Accepted: 11/26/2024] [Indexed: 01/11/2025]
Abstract
In preimplantation embryos, epiblast (EPI) fate specification from the inner cell mass is controlled by the segregation of NANOG and GATA6 expression. TEAD-YAP interaction is activated during EPI formation and is required for pluripotency factor expression. These events occur asynchronously with similar timing during EPI formation, and their relationship remains elusive. Here, we examined the relationship between NANOG-GATA6 and TEAD-YAP. The nuclear accumulation of YAP takes place only in EPI-specified cells, and a positive feedback loop operates between NANOG and TEAD-YAP. The effects of TEAD-YAP on SOX2 upregulation in EPI-specified cells are likely indirect. EPI fate specification also alters the response of Nanog, Sox2 and Cdx2 to TEAD-YAP. These results suggest that EPI-fate specification alters the transcriptional network from the morula-like to the EPI-specified state and activates TEAD-YAP to trigger a positive feedback loop with NANOG, which stabilizes the EPI fate. The coordinated occurrence of these processes in individual cells likely supports proper EPI formation under the condition of asynchronous EPI-fate specification.
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Affiliation(s)
- Naoki Hirono
- Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
| | - Masakazu Hashimoto
- Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
- Japan Science and Technology Agency, PRESTO, Kawaguchi, Saitama 332-0012, Japan
| | - Hiromi Shimojo
- Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
| | - Hiroshi Sasaki
- Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
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19
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Sakaloglou P, Lazaros L, Bouba I, Markoula S, Zikopoulos A, Drakaki E, Anagnostaki I, Potiris A, Stavros S, Gerede A, Domali E, Drakakis P, Tzavaras T, Georgiou I. LINE-1-Induced Retrotransposition Affects Early Preimplantation Embryo DNA Integrity and Pluripotency. Int J Mol Sci 2024; 25:12722. [PMID: 39684434 DOI: 10.3390/ijms252312722] [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: 10/04/2024] [Revised: 10/28/2024] [Accepted: 11/24/2024] [Indexed: 12/18/2024] Open
Abstract
Retrotransposable elements are implicated in genome rearrangements and gene expression alterations that result in various human disorders. In the current study, we sought to investigate the potential effects of long interspersed elements-1 (LINE-1) overexpression on the integrity and methylation of DNA and on the expression of three major pluripotency factors (OCT4, SOX2, NANOG) during the preimplantation stages of human embryo development. Human MI oocytes were matured in vitro to MII and transfected through intracytoplasmic sperm injection (ICSI) either with an EGFP vector carrying a cloned active human LINE-1 retroelement or with the same EGFP vector without insert as control. The occurrence of retrotransposition events was screened by fluorescent microscopy. The in vitro preimplantation development as well as the methylation, pluripotency, and DNA double-strand breaks (DSBs) of the transfected embryos were examined. LINE-1 retrotransposons gave rise to new retrotransposition events in the transfected embryos. LINE-1 injected embryos were characterized by accelerated asymmetrical cell division, multiple cellular fragments, cleavage arrest, and degeneration. Early OCT4 expression remained unaltered, but cleavage arrest and a high fragmentation rate hindered the expression of SOX2/NANOG at the morula stage. Increased DNA DSBs were observed in cleavage-stage blastomeres, while no methylation changes were detected before the cleavage arrest. Our data provide evidence that LINE-1 retrotransposition in human preimplantation embryos may induce DNA DSBs, while at the same time, it appears to interfere with the expression patterns of pluripotency factors. The morphological, structural, and cleavage abnormalities of the transfected embryos show that aberrant retroelement expression may negatively affect human embryo development.
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Affiliation(s)
- Prodromos Sakaloglou
- Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
| | - Leandros Lazaros
- Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
- Medical Genetics and Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Ioannina University Hospital, 451 10 Ioannina, Greece
| | - Ioanna Bouba
- Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
| | - Sofia Markoula
- Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
| | - Athanasios Zikopoulos
- Third Department of Obstetrics and Gynecology, University General Hospital "ATTIKON", Medical School, National and Kapodistrian University of Athens, 124 62 Athens, Greece
| | - Eirini Drakaki
- First Department of Obstetrics and Gynecology, Alexandra Hospital, Medical School, National and Kapodistrian University of Athens, 115 28 Athens, Greece
| | - Ismini Anagnostaki
- Third Department of Obstetrics and Gynecology, University General Hospital "ATTIKON", Medical School, National and Kapodistrian University of Athens, 124 62 Athens, Greece
| | - Anastasios Potiris
- Third Department of Obstetrics and Gynecology, University General Hospital "ATTIKON", Medical School, National and Kapodistrian University of Athens, 124 62 Athens, Greece
| | - Sofoklis Stavros
- Third Department of Obstetrics and Gynecology, University General Hospital "ATTIKON", Medical School, National and Kapodistrian University of Athens, 124 62 Athens, Greece
| | - Angeliki Gerede
- Department of Obstetrics and Gynecology, Democritus University of Thrace, 691 00 Alexandroupolis, Greece
| | - Ekaterini Domali
- First Department of Obstetrics and Gynecology, Alexandra Hospital, Medical School, National and Kapodistrian University of Athens, 115 28 Athens, Greece
| | - Peter Drakakis
- Third Department of Obstetrics and Gynecology, University General Hospital "ATTIKON", Medical School, National and Kapodistrian University of Athens, 124 62 Athens, Greece
| | - Theodoros Tzavaras
- Department of General Biology, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
| | - Ioannis Georgiou
- Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece
- Medical Genetics and Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Ioannina University Hospital, 451 10 Ioannina, Greece
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20
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Holman AR, Tran S, Destici E, Farah EN, Li T, Nelson AC, Engler AJ, Chi NC. Single-cell multi-modal integrative analyses highlight functional dynamic gene regulatory networks directing human cardiac development. CELL GENOMICS 2024; 4:100680. [PMID: 39437788 PMCID: PMC11605693 DOI: 10.1016/j.xgen.2024.100680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 08/01/2024] [Accepted: 09/23/2024] [Indexed: 10/25/2024]
Abstract
Illuminating the precise stepwise genetic programs directing cardiac development provides insights into the mechanisms of congenital heart disease and strategies for cardiac regenerative therapies. Here, we integrate in vitro and in vivo human single-cell multi-omic studies with high-throughput functional genomic screening to reveal dynamic, cardiac-specific gene regulatory networks (GRNs) and transcriptional regulators during human cardiomyocyte development. Interrogating developmental trajectories reconstructed from single-cell data unexpectedly reveal divergent cardiomyocyte lineages with distinct gene programs based on developmental signaling pathways. High-throughput functional genomic screens identify key transcription factors from inferred GRNs that are functionally relevant for cardiomyocyte lineages derived from each pathway. Notably, we discover a critical heat shock transcription factor 1 (HSF1)-mediated cardiometabolic GRN controlling cardiac mitochondrial/metabolic function and cell survival, also observed in fetal human cardiomyocytes. Overall, these multi-modal genomic studies enable the systematic discovery and validation of coordinated GRNs and transcriptional regulators controlling the development of distinct human cardiomyocyte populations.
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Affiliation(s)
- Alyssa R Holman
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Shaina Tran
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Eugin Destici
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Elie N Farah
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Ting Li
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Aileena C Nelson
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
| | - Adam J Engler
- Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Engineering Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Sanford Consortium for Regenerative Medicine, La Jolla, CA 92093, USA
| | - Neil C Chi
- Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Engineering Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
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21
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Leme LDO, Carvalho JO, Mendes CM, Assumpção MEOD, Caetano AR, Franco MM, Dode MAN. Impact of sperm sex sorting on sperm quality and in vitro embryo production in bovine. Anim Reprod Sci 2024; 270:107604. [PMID: 39332062 DOI: 10.1016/j.anireprosci.2024.107604] [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: 03/07/2024] [Revised: 09/12/2024] [Accepted: 09/16/2024] [Indexed: 09/29/2024]
Abstract
Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen - CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.
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Affiliation(s)
- Ligiane de Oliveira Leme
- UFES, Universidade Federal do Espírito Santo - Alto Universitario, S/N - Guararema, Alegre, ES 29500-000, Brazil
| | - José Oliveira Carvalho
- UFES, Universidade Federal do Espírito Santo - Alto Universitario, S/N - Guararema, Alegre, ES 29500-000, Brazil
| | - Camilla Motta Mendes
- USP, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87 - Butantã, São Paulo, SP 05508-900, Brazil
| | | | - Alexandre Rodrigues Caetano
- Embrapa Recursos Genéticos e Biotecnologia - Parque Estação Biológica, PqEB s/nº Av. W5 Norte (final) - PO Box 02372, Brasília, DF 70770-917, Brazil
| | - Maurício Machaim Franco
- Embrapa Recursos Genéticos e Biotecnologia - Parque Estação Biológica, PqEB s/nº Av. W5 Norte (final) - PO Box 02372, Brasília, DF 70770-917, Brazil
| | - Margot Alves Nunes Dode
- Embrapa Recursos Genéticos e Biotecnologia - Parque Estação Biológica, PqEB s/nº Av. W5 Norte (final) - PO Box 02372, Brasília, DF 70770-917, Brazil.
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22
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Rosen BP, Li QV, Cho HS, Liu D, Yang D, Graff S, Yan J, Luo R, Verma N, Damodaran JR, Kale HT, Kaplan SJ, Beer MA, Sidoli S, Huangfu D. Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness. Nat Commun 2024; 15:8966. [PMID: 39419994 PMCID: PMC11487130 DOI: 10.1038/s41467-024-53284-4] [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: 08/25/2023] [Accepted: 10/08/2024] [Indexed: 10/19/2024] Open
Abstract
Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.
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Affiliation(s)
- Bess P Rosen
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA
| | - Qing V Li
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Tessera Therapeutics, Somerville, MA, USA
| | - Hyein S Cho
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
| | - Dingyu Liu
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Dapeng Yang
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
| | - Sarah Graff
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Jielin Yan
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Renhe Luo
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Nipun Verma
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA
| | | | - Hanuman T Kale
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
| | - Samuel J Kaplan
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA
| | - Michael A Beer
- Department of Biomedical Engineering and McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA
| | - Simone Sidoli
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Danwei Huangfu
- Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA.
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23
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Liu T, Shen X, Ren Y, Lu H, Liu Y, Chen C, Yu L, Xue Z. Genome-wide mapping of native co-localized G4s and R-loops in living cells. eLife 2024; 13:RP99026. [PMID: 39392462 PMCID: PMC11469684 DOI: 10.7554/elife.99026] [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] [Indexed: 10/12/2024] Open
Abstract
The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease.
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Affiliation(s)
- Ting Liu
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
| | - Xing Shen
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
| | - Yijia Ren
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
| | - Hongyu Lu
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
| | - Yu Liu
- Department of Hematology and Institute of Hematology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan UniversityChengduChina
| | - Chong Chen
- Department of Hematology and Institute of Hematology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan UniversityChengduChina
| | - Lin Yu
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
| | - Zhihong Xue
- Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan UniversityChengduChina
- Development and Related Diseases of Women and Children Key Laboratory of Sichuan ProvinceChengduChina
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24
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Festuccia N, Vandormael-Pournin S, Chervova A, Geiselmann A, Langa-Vives F, Coux RX, Gonzalez I, Collet GG, Cohen-Tannoudji M, Navarro P. Nr5a2 is dispensable for zygotic genome activation but essential for morula development. Science 2024; 386:eadg7325. [PMID: 39361745 DOI: 10.1126/science.adg7325] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2023] [Revised: 05/10/2024] [Accepted: 08/05/2024] [Indexed: 10/05/2024]
Abstract
Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.
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Affiliation(s)
- Nicola Festuccia
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Sandrine Vandormael-Pournin
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Almira Chervova
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Anna Geiselmann
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
- Sorbonne Université, Complexité du Vivant, 75005 Paris, France
| | | | - Rémi-Xavier Coux
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Inma Gonzalez
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Guillaume Giraud Collet
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
- Université Paris Cité, BioSPC, 75013 Paris, France
| | - Michel Cohen-Tannoudji
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
| | - Pablo Navarro
- Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Epigenomics, Proliferation, and the Identity of Cells Unit, 75015 Paris, France
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25
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SoRelle ED, Haynes LE, Willard KA, Chang B, Ch’ng J, Christofk H, Luftig MA. Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression. PLoS Pathog 2024; 20:e1012341. [PMID: 39446925 PMCID: PMC11563402 DOI: 10.1371/journal.ppat.1012341] [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: 06/17/2024] [Revised: 11/14/2024] [Accepted: 10/02/2024] [Indexed: 10/26/2024] Open
Abstract
Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in three B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.
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Affiliation(s)
- Elliott D. SoRelle
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Duke Center for Virology, Durham, North Carolina, United States of America
| | - Lauren E. Haynes
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Duke Center for Virology, Durham, North Carolina, United States of America
| | - Katherine A. Willard
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Duke Center for Virology, Durham, North Carolina, United States of America
| | - Beth Chang
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, North Carolina, United States of America
| | - James Ch’ng
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California, United States of America
| | - Heather Christofk
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California, United States of America
- Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California, United States of America
| | - Micah A. Luftig
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Duke Center for Virology, Durham, North Carolina, United States of America
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26
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Wu Z, Ma X, Wang J. NCOA3 knockdown delays human embryo development. Heliyon 2024; 10:e37639. [PMID: 39315150 PMCID: PMC11417216 DOI: 10.1016/j.heliyon.2024.e37639] [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: 05/05/2024] [Revised: 08/17/2024] [Accepted: 09/06/2024] [Indexed: 09/25/2024] Open
Abstract
Embryonic development is a precisely controlled sequential process influenced by complex external and internal factors; therefore, this process holds paramount significance in the context of in vitro fertilization and embryo transfer (IVF-ET), with internal oocyte and embryo quality being pivotal in determining its success. Nuclear receptor coactivator 3 (NCOA3), a member of the p160 nuclear receptor coactivators family, has been extensively studied in tumorigenesis and reportedly plays a crucial role in maintaining pluripotency in mouse embryonic stem cells (ESCs). However, its functions in human embryo development remain largely unexplored. In this study, we collected human samples, including oocytes, zygotes, and embryos, from patients at the First Affiliated Hospital of Zhengzhou University to investigate whether NCOA3 regulates human embryonic development. To this end, we employed various assays, including immunofluorescence, quantitative real-time PCR (qPCR), microinjection, and RNA sequencing. Our findings suggested that NCOA3 expression level was low in inferior embryos (with >50 % fragmentation), and its presence is closely related to the expression of the pluripotency factor NANOG. Deletion of NCOA3 delays human embryonic development. Single-oocyte RNA sequencing revealed that NCOA3 primarily participates in metabolic alterations in oocytes. In sum, these findings elucidate the pivotal roles of NCOA3 in human embryonic development-NCOA3 deletion compromise the developmental potential of embryos. These mechanistic insights into the role of NCOA3 in human embryonic development not only advances our understanding of the intricate molecular mechanisms involved but also holds potential implications for improving assisted reproductive technologies (ART) and addressing developmental disorders in human embryos.
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Affiliation(s)
- Zhaoting Wu
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, China
| | - Xueshan Ma
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, China
- Department of Reproduction and Genetics, The Affiliated Taian City Central Hospital of Qingdao University, Taian, 271000, China
| | - Jingyu Wang
- School of Biomedical Engineering and Technology, Tianjin Medical University, Tianjin, 300070, China
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27
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Qian Y, Ye Y, Zhang W, Wu Q. Npac Regulates Pre-mRNA Splicing in Mouse Embryonic Stem Cells. Int J Mol Sci 2024; 25:10396. [PMID: 39408725 PMCID: PMC11477393 DOI: 10.3390/ijms251910396] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2024] [Revised: 09/22/2024] [Accepted: 09/24/2024] [Indexed: 10/20/2024] Open
Abstract
As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.
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Affiliation(s)
- Yiwei Qian
- Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, China;
- State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, China
| | - Ying Ye
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou 215123, China
| | - Wensheng Zhang
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou 215123, China
| | - Qiang Wu
- State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, China
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28
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Yu M, Wang F, Gang H, Liu C. Research progress of nanog gene in fish. Mol Genet Genomics 2024; 299:88. [PMID: 39313603 DOI: 10.1007/s00438-024-02182-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2024] [Accepted: 09/07/2024] [Indexed: 09/25/2024]
Abstract
Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.
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Affiliation(s)
- Miao Yu
- Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Engineering Laboratory of Henan Province for Aquatic Animal Disease Control, Observation and Research Station On Water Ecosystem in Danjiangkou Reservoir of Henan Province, College of Fisheries, Henan Normal University, Xinxiang, 453007, China.
| | - Fangyuan Wang
- Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Engineering Laboratory of Henan Province for Aquatic Animal Disease Control, Observation and Research Station On Water Ecosystem in Danjiangkou Reservoir of Henan Province, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Huihui Gang
- Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Engineering Laboratory of Henan Province for Aquatic Animal Disease Control, Observation and Research Station On Water Ecosystem in Danjiangkou Reservoir of Henan Province, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Chuanhu Liu
- School of 3D Printing, Xinxiang University, Xinxiang, 453003, China.
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29
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Lee SE, Lee HB, Yoon JW, Park HJ, Kim SH, Han DH, Lim ES, Kim EY, Park SP. Rapamycin treatment during prolonged in vitro maturation enhances the developmental competence of immature porcine oocytes. JOURNAL OF ANIMAL SCIENCE AND TECHNOLOGY 2024; 66:905-919. [PMID: 39398303 PMCID: PMC11466741 DOI: 10.5187/jast.2023.e101] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/25/2023] [Revised: 08/25/2023] [Accepted: 09/23/2023] [Indexed: 10/15/2024]
Abstract
Porcine oocytes undergo in vitro maturation (IVM) for 42-44 h. During this period, most oocytes proceed to metaphase and then to pro-metaphase if the nucleus has sufficiently matured. Forty-four hours is sufficient for oocyte nuclear maturation but not for full maturation of the oocyte cytoplasm. This study investigated the influences of extension of the IVM duration with rapamycin treatment on molecular maturation factors. The phospho-p44/42 mitogen-activated protein kinase (MAPK) level was enhanced in comparison with the total p44/42 MAPK level after 52 h of IVM. Oocytes were treated with and without 10 μM rapamycin (10 R and 0 R, respectively) and examined after 52 h of IVM, whereas control oocytes were examined after 44 h of IVM. Phospho-p44/42 MAPK activity was upregulated the 10 R and 0 R oocytes than in control oocytes. The expression levels of maternal genes were highest in 10 R oocytes and were higher in 0 R oocytes than in control oocytes. Reactive oxygen species (ROS) activity was dramatically increased in 0 R oocytes but was similar in 10 R and control oocytes. The 10 R group exhibited an increased embryo development rate, a higher total cell number per blastocyst, and decreased DNA fragmentation. The mRNA level of development-related (POU5F1 and NANOG) mRNA, oocyte-apoptotic (BCL2L1) genes were highest in 10 R blastocysts. These results suggest that prolonged IVM duration with rapamycin treatment represses ROS production and increases expression of molecular maturation factors. Therefore, this is a good strategy to enhance the developmental capacity in porcine oocytes.
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Affiliation(s)
- Seung-Eun Lee
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Faculty of Biotechnology, College of
Applied Life Sciences, Jeju National University, Jeju 63243,
Korea
- Subtropical Livestock Research Institute,
National Institute of Animal Science, RDA, Jeju 63242,
Korea
| | - Han-Bi Lee
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Faculty of Biotechnology, College of
Applied Life Sciences, Jeju National University, Jeju 63243,
Korea
| | - Jae-Wook Yoon
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
| | - Hyo-Jin Park
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
| | - So-Hee Kim
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
| | - Dong-Hun Han
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Faculty of Biotechnology, College of
Applied Life Sciences, Jeju National University, Jeju 63243,
Korea
| | - Eun-Seo Lim
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Faculty of Biotechnology, College of
Applied Life Sciences, Jeju National University, Jeju 63243,
Korea
| | - Eun-Young Kim
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Faculty of Biotechnology, College of
Applied Life Sciences, Jeju National University, Jeju 63243,
Korea
- Mirae Cell Bio, Seoul 04795,
Korea
| | - Se-Pill Park
- Stem Cell Research Center, Jeju National
University, Jeju 63243, Korea
- Mirae Cell Bio, Seoul 04795,
Korea
- Department of Bio Medical Informatic,
College of Applied Life Sciences, Jeju National University,
Jeju 63242, Korea
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30
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Rosen BP, Li QV, Cho HS, Liu D, Yang D, Graff S, Yan J, Luo R, Verma N, Damodaran JR, Kale HT, Kaplan SJ, Beer MA, Sidoli S, Huangfu D. Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.03.539283. [PMID: 37205540 PMCID: PMC10187244 DOI: 10.1101/2023.05.03.539283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.
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31
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Alowaysi M, Baadhaim M, Al-Shehri M, Alzahrani H, Badkok A, Attas H, Zakri S, Alameer S, Malibari D, Hosawi M, Daghestani M, Al-Ghamdi K, Muharraq M, Zia A, Tegne J, Alfadhel M, Aboalola D, Alsayegh K. Derivation of two iPSC lines (KAIMRCi004-A, KAIMRCi004-B) from a Saudi patient with Biotin-Thiamine-responsive Basal Ganglia Disease (BTBGD) carrying homozygous pathogenic missense variant in the SCL19A3 gene. Hum Cell 2024; 37:1567-1577. [PMID: 38980565 PMCID: PMC11341592 DOI: 10.1007/s13577-024-01097-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2024] [Accepted: 06/27/2024] [Indexed: 07/10/2024]
Abstract
The neurometabolic disorder known as biotin-thiamine-responsive basal ganglia disease (BTBGD) is a rare autosomal recessive condition linked to bi-allelic pathogenic mutations in the SLC19A3 gene. BTBGD is characterized by progressive encephalopathy, confusion, seizures, dysarthria, dystonia, and severe disabilities. Diagnosis is difficult due to the disease's rare nature and diverse clinical characteristics. The primary treatment for BTBGD at this time is thiamine and biotin supplementation, while its long-term effectiveness is still being investigated. In this study, we have generated two clones of induced pluripotent stem cells (iPSCs) from a 10-year-old female BTBGD patient carrying a homozygous mutation for the pathogenic variant in exon 5 of the SLC19A3 gene, c.1264A > G (p.Thr422Ala). We have confirmed the pluripotency of the generated iPS lines and successfully differentiated them to neural progenitors. Because our understanding of genotype-phenotype correlations in BTBGD is limited, the establishment of BTBGD-iPSC lines with a homozygous SLC19A3 mutation provides a valuable cellular model to explore the molecular mechanisms underlying SLC19A3-associated cellular dysfunction. This model holds potential for advancing the development of novel therapeutic strategies.
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Affiliation(s)
- Maryam Alowaysi
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Moayad Baadhaim
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Mohammad Al-Shehri
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Hajar Alzahrani
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Amani Badkok
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Hanouf Attas
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Samer Zakri
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Seham Alameer
- Clinical Biomedical Genetics, Ministry of the National Guard Health Affairs, Jeddah, Saudi Arabia
| | - Dalal Malibari
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Manal Hosawi
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
| | - Mustafa Daghestani
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia
- Department of Pathology and Laboratory Medicine, Ministry of the National Guard Health Affairs, Jeddah, Saudi Arabia
| | - Khalid Al-Ghamdi
- Forensic Laboratories, Criminal Evidence Department, Jeddah, Saudi Arabia
| | - Mohammed Muharraq
- Forensic Laboratories, Criminal Evidence Department, Jeddah, Saudi Arabia
| | - Asima Zia
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
| | - Jesper Tegne
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
- Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
| | - Majid Alfadhel
- Department of Genetics and Precision Medicine, King Abdullah Specialized Children Hospital, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia
- Department of Medical Genomics Research, King Abdullah International Medical Research Center, Ministry of National Guard Health Affairs, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
| | - Doaa Aboalola
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia.
| | - Khaled Alsayegh
- King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah, Saudi Arabia.
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32
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Skory RM. Revisiting trophectoderm-inner cell mass lineage segregation in the mammalian preimplantation embryo. Hum Reprod 2024; 39:1889-1898. [PMID: 38926157 PMCID: PMC12102071 DOI: 10.1093/humrep/deae142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 06/06/2024] [Indexed: 06/28/2024] Open
Abstract
In the first days of life, cells of the mammalian embryo segregate into two distinct lineages, trophectoderm and inner cell mass. Unlike nonmammalian species, mammalian development does not proceed from predetermined factors in the oocyte. Rather, asymmetries arise de novo in the early embryo incorporating cues from cell position, contractility, polarity, and cell-cell contacts. Molecular heterogeneities, including transcripts and non-coding RNAs, have now been characterized as early as the 2-cell stage. However, it's debated whether these early heterogeneities bias cells toward one fate or the other or whether lineage identity arises stochastically at the 16-cell stage. This review summarizes what is known about early blastomere asymmetries and our understanding of lineage allocation in the context of historical models. Preimplantation development is reviewed coupled with what is known about changes in morphology, contractility, and transcription factor networks. The addition of single-cell atlases of human embryos has begun to reveal key differences between human and mouse, including the timing of events and core transcription factors. Furthermore, the recent generation of blastoid models will provide valuable tools to test and understand fate determinants. Lastly, new techniques are reviewed, which may better synthesize existing knowledge with emerging data sets and reconcile models with the regulative capacity unique to the mammalian embryo.
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Affiliation(s)
- Robin M Skory
- Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Cell and Developmental Biology, Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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33
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Mohajer F, Khoradmehr A, Riazalhosseini B, Zendehboudi T, Nabipour I, Baghban N. In vitro detection of marine invertebrate stem cells: utilizing molecular and cellular biology techniques and exploring markers. Front Cell Dev Biol 2024; 12:1440091. [PMID: 39239558 PMCID: PMC11374967 DOI: 10.3389/fcell.2024.1440091] [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: 05/28/2024] [Accepted: 08/07/2024] [Indexed: 09/07/2024] Open
Abstract
Marine invertebrate stem cells (MISCs) represent a distinct category of pluripotent and totipotent cells with remarkable abilities for self-renewal and differentiation into multiple germ layers, akin to their vertebrate counterparts. These unique cells persist throughout an organism's adult life and have been observed in various adult marine invertebrate phyla. MISCs play crucial roles in numerous biological processes, including developmental biology phenomena specific to marine invertebrates, such as senescence, delayed senescence, whole-body regeneration, and asexual reproduction. Furthermore, they serve as valuable models for studying stem cell biology. Despite their significance, information about MISCs remains scarce and scattered in the scientific literature. In this review, we have carefully collected and summarized valuable information about MISC detection by perusing the articles that study and detect MISCs in various marine invertebrate organisms. The review begins by defining MISCs and highlighting their unique features compared to vertebrates. It then discusses the common markers for MISC detection and in vitro techniques employed in invertebrate and vertebrates investigation. This comprehensive review provides researchers and scientists with a cohesive and succinct overview of MISC characteristics, detection methods, and associated biological phenomena in marine invertebrate organisms. We aim to offer a valuable resource to researchers and scientists interested in marine invertebrate stem cells, fostering a better understanding of their broader implications in biology. With ongoing advancements in scientific techniques and the continued exploration of marine invertebrate species, we anticipate that further discoveries will expand our knowledge of MISCs and their broader implications in biology.
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Affiliation(s)
- Fatemeh Mohajer
- Student Research and Technology Committee, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Arezoo Khoradmehr
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Behnaz Riazalhosseini
- The Pharmacogenomics Laboratory, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Tuba Zendehboudi
- Student Research and Technology Committee, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Iraj Nabipour
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Neda Baghban
- Food Control Laboratory, Food and Drug Deputy, Bushehr University of Medical Sciences, Bushehr, Iran
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34
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Ulfig A, Jakob U. Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions. Dev Cell 2024; 59:2118-2133.e8. [PMID: 39106861 PMCID: PMC11338707 DOI: 10.1016/j.devcel.2024.07.008] [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: 12/22/2023] [Revised: 04/23/2024] [Accepted: 07/09/2024] [Indexed: 08/09/2024]
Abstract
Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates.
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Affiliation(s)
- Agnes Ulfig
- Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
| | - Ursula Jakob
- Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA; Biological Chemistry Department, University of Michigan Medical School, Ann Arbor, MI, USA.
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35
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Su Q, Yang SP, Guo JP, Rong YR, Sun Y, Chai YR. Epigallocatechin-3-gallate ameliorates lipopolysaccharide-induced acute thymus involution in mice via AMPK/Sirt1 pathway. Microbiol Immunol 2024; 68:281-293. [PMID: 38886542 DOI: 10.1111/1348-0421.13159] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 05/08/2024] [Accepted: 05/27/2024] [Indexed: 06/20/2024]
Abstract
The thymus, a site to culture the naïve T lymphocytes, is susceptible to atrophy or involution due to aging, inflammation, and oxidation. Epigallocatechin-3-gallate (EGCG) has been proven to possess anti-inflammatory, antioxidant, and antitumor activity. Here, we investigate the effects of EGCG on thymic involution induced by lipopolysaccharide (LPS), an endotoxin derived from Gram-negative bacteria. The methodology included an in vivo experiment on female Kunming mice exposed to LPS and EGCG. Morphological assessment of thymic involution, immunohistochemical detection, and thymocyte subsets analysis by flow cytometry were further carried out to evaluate the potential role of EGCG on the thymus. As a result, we found that EGCG alleviated LPS-induced thymic atrophy, increased mitochondrial membrane potential and superoxide dismutase levels, and decreased malondialdehyde and reactive oxygen species levels. In addition, EGCG pre-supplement restored the ratio of thymocyte subsets, the expression of autoimmune regulator, sex-determining region Y-box 2, and Nanog homebox, and reduced the number of senescent cells and collagen fiber deposition. Western blotting results indicated that EGCG treatment elevated LPS-induced decrease in pAMPK, Sirt1 protein expression. Collectively, EGCG relieved thymus architecture and function damaged by LPS via regulation of AMPK/Sirt1 signaling pathway. Our findings may provide a new strategy on protection of thymus from involution caused by LPS by using EGCG. And EGCG might be considered as a potential agent for the prevention and treatment of thymic involution.
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Affiliation(s)
- Qing Su
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
- The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, PR China
| | - Shu-Ping Yang
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
- School of Medical Technology, Sanquan College of Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Jun-Ping Guo
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
| | - Yi-Ren Rong
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
| | - Yun Sun
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
| | - Yu-Rong Chai
- Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
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Dattani A, Corujo-Simon E, Radley A, Heydari T, Taheriabkenar Y, Carlisle F, Lin S, Liddle C, Mill J, Zandstra PW, Nichols J, Guo G. Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification. Cell Stem Cell 2024; 31:1058-1071.e5. [PMID: 38823388 DOI: 10.1016/j.stem.2024.05.003] [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: 12/05/2023] [Revised: 03/13/2024] [Accepted: 05/07/2024] [Indexed: 06/03/2024]
Abstract
The hypoblast is an essential extraembryonic tissue set aside within the inner cell mass in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naive pluripotent stem cell (PSC) to hypoblast differentiation proceeds via reversion to a transitional ICM-like state from which the hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signaling is critical for hypoblast specification. Revisiting FGF signaling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro, the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mice and humans. Overall, this study demonstrates the utility of human naive PSC-based models in elucidating the mechanistic features of early human embryogenesis.
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Affiliation(s)
- Anish Dattani
- Living Systems Institute, University of Exeter, Exeter, UK; Department of Clinical & Biomedical Sciences, University of Exeter Medical School, Faculty of Health and Life Sciences, University of Exeter, Exeter, UK
| | - Elena Corujo-Simon
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, UK
| | - Arthur Radley
- Living Systems Institute, University of Exeter, Exeter, UK
| | - Tiam Heydari
- Michael Smith Laboratories, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | | | | | - Simeng Lin
- Department of Clinical & Biomedical Sciences, University of Exeter Medical School, Faculty of Health and Life Sciences, University of Exeter, Exeter, UK
| | - Corin Liddle
- Bioimaging Centre, University of Exeter, Exeter, UK
| | - Jonathan Mill
- Department of Clinical & Biomedical Sciences, University of Exeter Medical School, Faculty of Health and Life Sciences, University of Exeter, Exeter, UK
| | - Peter W Zandstra
- Michael Smith Laboratories, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | - Jennifer Nichols
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, UK
| | - Ge Guo
- Living Systems Institute, University of Exeter, Exeter, UK; Department of Clinical & Biomedical Sciences, University of Exeter Medical School, Faculty of Health and Life Sciences, University of Exeter, Exeter, UK.
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SoRelle ED, Haynes LE, Willard KA, Chang B, Ch’ng J, Christofk H, Luftig MA. Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.14.598975. [PMID: 38915538 PMCID: PMC11195279 DOI: 10.1101/2024.06.14.598975] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/26/2024]
Abstract
Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in two B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.
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Affiliation(s)
- Elliott D. SoRelle
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Duke Center for Virology, Durham, NC 27710, USA
| | - Lauren E. Haynes
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Duke Center for Virology, Durham, NC 27710, USA
| | - Katherine A. Willard
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Duke Center for Virology, Durham, NC 27710, USA
| | - Beth Chang
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
| | - James Ch’ng
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
| | - Heather Christofk
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA
| | - Micah A. Luftig
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Duke Center for Virology, Durham, NC 27710, USA
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38
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Zhu M, Meglicki M, Lamba A, Wang P, Royer C, Turner K, Jauhar MA, Jones C, Child T, Coward K, Na J, Zernicka-Goetz M. Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification. Nat Struct Mol Biol 2024; 31:964-976. [PMID: 38789684 PMCID: PMC11189297 DOI: 10.1038/s41594-024-01311-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Accepted: 04/09/2024] [Indexed: 05/26/2024]
Abstract
The mouse and human embryo gradually loses totipotency before diversifying into the inner cell mass (ICM, future organism) and trophectoderm (TE, future placenta). The transcription factors TFAP2C and TEAD4 with activated RHOA accelerate embryo polarization. Here we show that these factors also accelerate the loss of totipotency. TFAP2C and TEAD4 paradoxically promote and inhibit Hippo signaling before lineage diversification: they drive expression of multiple Hippo regulators while also promoting apical domain formation, which inactivates Hippo. Each factor activates TE specifiers in bipotent cells, while TFAP2C also activates specifiers of the ICM fate. Asymmetric segregation of the apical domain reconciles the opposing regulation of Hippo signaling into Hippo OFF and the TE fate, or Hippo ON and the ICM fate. We propose that the bistable switch established by TFAP2C and TEAD4 is exploited to trigger robust lineage diversification in the developing embryo.
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Affiliation(s)
- Meng Zhu
- Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
- Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
| | - Maciej Meglicki
- Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | - Adiyant Lamba
- Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | - Peizhe Wang
- Centre for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, China
| | - Christophe Royer
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
| | - Karen Turner
- Oxford Fertility, Institute of Reproductive Sciences, Oxford, UK
| | - Muhammad Abdullah Jauhar
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
| | - Celine Jones
- Nuffield Department of Women's and Reproductive Health, Level 3, Women's Centre, John Radcliffe Hospital, University of Oxford, Oxford, UK
| | - Tim Child
- Nuffield Department of Women's and Reproductive Health, Level 3, Women's Centre, John Radcliffe Hospital, University of Oxford, Oxford, UK
| | - Kevin Coward
- Nuffield Department of Women's and Reproductive Health, Level 3, Women's Centre, John Radcliffe Hospital, University of Oxford, Oxford, UK
| | - Jie Na
- Centre for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, China
| | - Magdalena Zernicka-Goetz
- Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
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39
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Wang L, Yi S, Cui X, Guo Z, Wang M, Kou X, Zhao Y, Wang H, Jiang C, Gao S, Yang G, Chen J, Gao R. Chromatin landscape instructs precise transcription factor regulome during embryonic lineage specification. Cell Rep 2024; 43:114136. [PMID: 38643480 DOI: 10.1016/j.celrep.2024.114136] [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: 11/29/2023] [Revised: 03/10/2024] [Accepted: 04/08/2024] [Indexed: 04/23/2024] Open
Abstract
Embryos, originating from fertilized eggs, undergo continuous cell division and differentiation, accompanied by dramatic changes in transcription, translation, and metabolism. Chromatin regulators, including transcription factors (TFs), play indispensable roles in regulating these processes. Recently, the trophoblast regulator TFAP2C was identified as crucial in initiating early cell fate decisions. However, Tfap2c transcripts persist in both the inner cell mass and trophectoderm of blastocysts, prompting inquiry into Tfap2c's function in post-lineage establishment. In this study, we delineate the dynamics of TFAP2C during the mouse peri-implantation stage and elucidate its collaboration with the key lineage regulators CDX2 and NANOG. Importantly, we propose that de novo formation of H3K9me3 in the extraembryonic ectoderm during implantation antagonizes TFAP2C binding to crucial developmental genes, thereby maintaining its lineage identity. Together, these results highlight the plasticity of the chromatin environment in designating the genomic binding of highly adaptable lineage-specific TFs and regulating embryonic cell fates.
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Affiliation(s)
- Liping Wang
- Shanghai Tenth People's Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200072, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Shanru Yi
- Shanghai Tenth People's Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200072, China; Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Xinyu Cui
- Shanghai Tenth People's Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200072, China; Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of the Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200065, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Zhenxiang Guo
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Mengting Wang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Xiaochen Kou
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Yanhong Zhao
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Hong Wang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Cizhong Jiang
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of the Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200065, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China
| | - Shaorong Gao
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China; Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai East Hospital, Tongji University, Shanghai 200120, China.
| | - Guang Yang
- Shanghai Tenth People's Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200072, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China.
| | - Jiayu Chen
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China.
| | - Rui Gao
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China.
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40
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He J, Huo X, Pei G, Jia Z, Yan Y, Yu J, Qu H, Xie Y, Yuan J, Zheng Y, Hu Y, Shi M, You K, Li T, Ma T, Zhang MQ, Ding S, Li P, Li Y. Dual-role transcription factors stabilize intermediate expression levels. Cell 2024; 187:2746-2766.e25. [PMID: 38631355 DOI: 10.1016/j.cell.2024.03.023] [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: 06/09/2023] [Revised: 12/08/2023] [Accepted: 03/18/2024] [Indexed: 04/19/2024]
Abstract
Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.
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Affiliation(s)
- Jinnan He
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Xiangru Huo
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Gaofeng Pei
- State Key Laboratory of Membrane Biology, Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, China
| | - Zeran Jia
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Yiming Yan
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Jiawei Yu
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Haozhi Qu
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Yunxin Xie
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Junsong Yuan
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Yuan Zheng
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
| | - Yanyan Hu
- School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, China
| | - Minglei Shi
- Bioinformatics Division, National Research Center for Information Science and Technology, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Kaiqiang You
- Department of Biomedical Informatics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
| | - Tingting Li
- Department of Biomedical Informatics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
| | - Tianhua Ma
- School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, China
| | - Michael Q Zhang
- Bioinformatics Division, National Research Center for Information Science and Technology, School of Medicine, Tsinghua University, Beijing 100084, China; Department of Biological Sciences, Center for Systems Biology, The University of Texas, Dallas, TX 75080-3021, USA
| | - Sheng Ding
- School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, China
| | - Pilong Li
- State Key Laboratory of Membrane Biology, Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, China.
| | - Yinqing Li
- The IDG/McGovern Institute for Brain Research, MOE Key Laboratory of Bioinformatics, State Key Lab of Molecular Oncology, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China.
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41
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Ibrahim M, Grochowska E, Lázár B, Várkonyi E, Bednarczyk M, Stadnicka K. The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells. Genes (Basel) 2024; 15:624. [PMID: 38790253 PMCID: PMC11121574 DOI: 10.3390/genes15050624] [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: 04/15/2024] [Revised: 05/10/2024] [Accepted: 05/13/2024] [Indexed: 05/26/2024] Open
Abstract
Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.
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Affiliation(s)
- Mariam Ibrahim
- Department of Animal Biotechnology and Genetics, Bydgoszcz University of Science and Technology, Mazowiecka 28, 85-084 Bydgoszcz, Poland
- PBS Doctoral School, Bydgoszcz University of Science and Technology, Aleje Prof. S. Kaliskiego 7, 85-796 Bydgoszcz, Poland
| | - Ewa Grochowska
- Department of Animal Biotechnology and Genetics, Bydgoszcz University of Science and Technology, Mazowiecka 28, 85-084 Bydgoszcz, Poland
| | - Bence Lázár
- National Centre for Biodiversity and Gene Conservation, Institute for Farm Animal Gene Conservation, Isaszegi Street 200, 2100 Godollo, Hungary
- Institute of Genetics and Biotechnology, Hungarian University of Agriculture and Life Sciences, Szent-Gyorgyi Albert Street 4, 2100 Godollo, Hungary
| | - Eszter Várkonyi
- National Centre for Biodiversity and Gene Conservation, Institute for Farm Animal Gene Conservation, Isaszegi Street 200, 2100 Godollo, Hungary
| | - Marek Bednarczyk
- Department of Animal Biotechnology and Genetics, Bydgoszcz University of Science and Technology, Mazowiecka 28, 85-084 Bydgoszcz, Poland
| | - Katarzyna Stadnicka
- Faculty of Health Sciences, Collegium Medicum, Nicolaus Copernicus University, Łukasiewicza 1, 85-821 Bydgoszcz, Poland
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42
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Li-Bao L, Díaz-Díaz C, Raiola M, Sierra R, Temiño S, Moya FJ, Rodriguez-Perales S, Santos E, Giovinazzo G, Bleckwehl T, Rada-Iglesias Á, Spitz F, Torres M. Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression. Nat Commun 2024; 15:3931. [PMID: 38729993 PMCID: PMC11087473 DOI: 10.1038/s41467-024-48258-5] [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: 11/12/2022] [Accepted: 04/23/2024] [Indexed: 05/12/2024] Open
Abstract
MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
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Affiliation(s)
- Lin Li-Bao
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro Andaluz de Biología del Desarrollo (CABD), Sevilla, Spain
| | - Covadonga Díaz-Díaz
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Morena Raiola
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Rocío Sierra
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Susana Temiño
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Francisco J Moya
- Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Sandra Rodriguez-Perales
- Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Elisa Santos
- Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain
| | - Giovanna Giovinazzo
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
- Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain
| | - Tore Bleckwehl
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Institute of Experimental Medicine and Systems Biology, RWTH Aachen University, Aachen, Germany
| | - Álvaro Rada-Iglesias
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria, Santander, Spain
| | - Francois Spitz
- Department of Human Genetics, The University of Chicago, Chicago, IL, USA
- Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Miguel Torres
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain.
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43
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Yanagihara K, Hayashi Y, Liu Y, Yamaguchi T, Hemmi Y, Kokunugi M, Yamada KU, Fukumoto K, Suga M, Terada S, Nikawa H, Kawabata K, Furue M. Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells. In Vitro Cell Dev Biol Anim 2024; 60:521-534. [PMID: 38169039 PMCID: PMC11126453 DOI: 10.1007/s11626-023-00824-9] [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: 08/12/2023] [Accepted: 09/08/2023] [Indexed: 01/05/2024]
Abstract
Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.
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Affiliation(s)
- Kana Yanagihara
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yohei Hayashi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Yujung Liu
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Tomoko Yamaguchi
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yasuko Hemmi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan
| | - Minako Kokunugi
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kozue Uchio Yamada
- Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Ken Fukumoto
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Mika Suga
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Satoshi Terada
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Hiroki Nikawa
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kenji Kawabata
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Miho Furue
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan.
- Cel-MiM, Ltd., Tokyo, Japan.
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44
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Khan AQ, Hasan A, Mir SS, Rashid K, Uddin S, Steinhoff M. Exploiting transcription factors to target EMT and cancer stem cells for tumor modulation and therapy. Semin Cancer Biol 2024; 100:1-16. [PMID: 38503384 DOI: 10.1016/j.semcancer.2024.03.002] [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: 12/20/2023] [Revised: 03/15/2024] [Accepted: 03/15/2024] [Indexed: 03/21/2024]
Abstract
Transcription factors (TFs) are essential in controlling gene regulatory networks that determine cellular fate during embryogenesis and tumor development. TFs are the major players in promoting cancer stemness by regulating the function of cancer stem cells (CSCs). Understanding how TFs interact with their downstream targets for determining cell fate during embryogenesis and tumor development is a critical area of research. CSCs are increasingly recognized for their significance in tumorigenesis and patient prognosis, as they play a significant role in cancer initiation, progression, metastasis, and treatment resistance. However, traditional therapies have limited effectiveness in eliminating this subset of cells, allowing CSCs to persist and potentially form secondary tumors. Recent studies have revealed that cancer cells and tumors with CSC-like features also exhibit genes related to the epithelial-to-mesenchymal transition (EMT). EMT-associated transcription factors (EMT-TFs) like TWIST and Snail/Slug can upregulate EMT-related genes and reprogram cancer cells into a stem-like phenotype. Importantly, the regulation of EMT-TFs, particularly through post-translational modifications (PTMs), plays a significant role in cancer metastasis and the acquisition of stem cell-like features. PTMs, including phosphorylation, ubiquitination, and SUMOylation, can alter the stability, localization, and activity of EMT-TFs, thereby modulating their ability to drive EMT and stemness properties in cancer cells. Although targeting EMT-TFs holds potential in tackling CSCs, current pharmacological approaches to do so directly are unavailable. Therefore, this review aims to explore the role of EMT- and CSC-TFs, their connection and impact in cellular development and cancer, emphasizing the potential of TF networks as targets for therapeutic intervention.
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Affiliation(s)
- Abdul Q Khan
- Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar.
| | - Adria Hasan
- Molecular Cell Biology Laboratory, Integral Information and Research Centre-4 (IIRC-4), Integral University, Kursi Road, Lucknow 226026, India; Department of Bioengineering, Faculty of Engineering, Integral University, Kursi Road, Lucknow 226026, India
| | - Snober S Mir
- Molecular Cell Biology Laboratory, Integral Information and Research Centre-4 (IIRC-4), Integral University, Kursi Road, Lucknow 226026, India; Department of Biosciences, Faculty of Science, Integral University, Kursi Road, Lucknow 226026, India
| | - Khalid Rashid
- Department of Urology,Feinberg School of Medicine, Northwestern University, 303 E Superior Street, Chicago, IL 60611, USA
| | - Shahab Uddin
- Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar; Department of Biosciences, Faculty of Science, Integral University, Kursi Road, Lucknow 226026, India; Laboratory Animal Research Center, Qatar University, Doha, Qatar; Dermatology Institute, Academic Health System, Hamad Medical Corporation, Doha 3050, Qatar
| | - Martin Steinhoff
- Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar; Dermatology Institute, Academic Health System, Hamad Medical Corporation, Doha 3050, Qatar; Department of Dermatology and Venereology, Rumailah Hospital, Hamad Medical Corporation, Doha 3050, Qatar; Department of Medicine, Weill Cornell Medicine Qatar, Qatar Foundation-Education City, Doha 24144, Qatar; Department of Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA; College of Medicine, Qatar University, Doha 2713, Qatar
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45
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Jin SW, Seong Y, Yoon D, Kwon YS, Song H. Dissolution of ribonucleoprotein condensates by the embryonic stem cell protein L1TD1. Nucleic Acids Res 2024; 52:3310-3326. [PMID: 38165001 PMCID: PMC11014241 DOI: 10.1093/nar/gkad1244] [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: 04/24/2023] [Revised: 11/22/2023] [Accepted: 12/18/2023] [Indexed: 01/03/2024] Open
Abstract
L1TD1 is a cytoplasmic RNA-binding protein specifically expressed in pluripotent stem cells and, unlike its mouse ortholog, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known protein-coding gene domesticated from a LINE-1 (L1) retroelement, the functional legacy of its ancestral protein, ORF1p of L1, and how it is manifested in L1TD1 are still unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs and localizes to high-density ribonucleoprotein (RNP) condensates. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. L1TD1 depletion promoted the formation of stress granules in embryonic stem cells. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain of L1TD1 facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatment of neurodegenerative diseases caused by disturbed stress granule dynamics.
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Affiliation(s)
- Sang Woo Jin
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea
| | - Youngmo Seong
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea
| | - Dayoung Yoon
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea
| | - Young-Soo Kwon
- Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul 05006, Republic of Korea
| | - Hoseok Song
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea
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46
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Giri A, Kar S. Interlinked bi-stable switches govern the cell fate commitment of embryonic stem cells. FEBS Lett 2024; 598:915-934. [PMID: 38408774 DOI: 10.1002/1873-3468.14832] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Revised: 12/23/2023] [Accepted: 02/03/2024] [Indexed: 02/28/2024]
Abstract
The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.
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Affiliation(s)
- Amitava Giri
- Department of Chemistry, IIT Bombay, Powai, India
| | - Sandip Kar
- Department of Chemistry, IIT Bombay, Powai, India
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47
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Mylonas KS, Peroulis M, Kapetanakis EI, Kapelouzou A. Myocardial Expression of Pluripotency, Longevity, and Proinflammatory Genes in the Context of Hypercholesterolemia and Statin Treatment. J Clin Med 2024; 13:1994. [PMID: 38610757 PMCID: PMC11012955 DOI: 10.3390/jcm13071994] [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: 01/31/2024] [Revised: 03/06/2024] [Accepted: 03/26/2024] [Indexed: 04/14/2024] Open
Abstract
Background: This study sought to assess the effect of statin therapy on myocardial inflammation in a White New Zealand rabbit model of atherogenesis. Methods: The mRNA expression levels of pro-inflammatory, pluripotency, and aging-related markers were quantified following a controlled feeding protocol and statin treatments. Results: Following high-cholesterol diet induction, we observed significant upregulation in the myocardial mRNA levels of MYD88, NF-κB, chemokines (CCL4, CCL20, and CCR2), IFN-γ, interleukins (IL-1β, IL-2, IL-4, IL-8, IL-10, and IL-18), and novel markers (klotho, KFL4, NANOG, and HIF1α). In contrast, HOXA5 expression was diminished following a hyperlipidemic diet. Both statin treatments significantly influenced the markers studied. Nevertheless, rosuvastatin administration resulted in a greater reduction in MYD88, NF-kB, chemokines (CCL4, CCL20, and CCR2), and interleukins IL-1β, IL-8, KLF4, NANOG, and HIF1α than fluvastatin. Fluvastatin, on the other hand, led to a stronger decrease in IL-4. Downregulation of IL-2 and IL-18 and upregulation of IFNβ and HOXA5 were comparable between the two statins. Notably, rosuvastatin had a stronger effect on the upregulation of klotho and IL-10. Conclusion: Overall, statin therapy significantly attenuated inflammatory, pluripotency, and klotho expression in myocardial tissue under atherogenic conditions. Our findings also highlight the differential efficacy of rosuvastatin over fluvastatin in curtailing proatherogenic inflammation, which could have profound implications for the clinical management of cardiovascular disease.
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Affiliation(s)
- Konstantinos S Mylonas
- Department of Cardiac Surgery, Onassis Cardiac Surgery Center, 356 Leof. Andreas Syngros, 17674 Athens, Greece
| | - Michail Peroulis
- Vascular Surgery Unit, Department of Surgery, Faculty of Medicine, University of Ioannina, 45110 Ioannina, Greece
| | - Emmanouil I Kapetanakis
- Third Department of Surgery, Attikon University Hospital, National and Kapodistrian University of Athens, 12462 Athens, Greece
| | - Alkistis Kapelouzou
- Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation Academy of Athens, 11527 Athens, Greece
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48
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Chea S, Kreger J, Lopez-Burks ME, MacLean AL, Lander AD, Calof AL. Gastrulation-stage gene expression in Nipbl+/- mouse embryos foreshadows the development of syndromic birth defects. SCIENCE ADVANCES 2024; 10:eadl4239. [PMID: 38507484 PMCID: PMC10954218 DOI: 10.1126/sciadv.adl4239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/19/2023] [Accepted: 02/15/2024] [Indexed: 03/22/2024]
Abstract
In animal models, Nipbl deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is Nipbl haploinsufficiency. Previous studies in Nipbl+/- mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and Nipbl+/- mouse embryos at gastrulation and early cardiac crescent stages. Nipbl+/- embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, Nanog was found to be overexpressed in all germ layers, and many gene expression changes observed in Nipbl+/- embryos could be attributed to Nanog overexpression. These findings establish a link between Nipbl deficiency, Nanog overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in Nipbl+/- animals and Cornelia de Lange syndrome.
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Affiliation(s)
- Stephenson Chea
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California Irvine, Irvine, CA 92697, USA
- Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697, USA
| | - Jesse Kreger
- Department of Quantitative and Computational Biology, Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA 90089, USA
| | - Martha E. Lopez-Burks
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California Irvine, Irvine, CA 92697, USA
- Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697, USA
| | - Adam L. MacLean
- Department of Quantitative and Computational Biology, Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA 90089, USA
| | - Arthur D. Lander
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California Irvine, Irvine, CA 92697, USA
- Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697, USA
| | - Anne L. Calof
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California Irvine, Irvine, CA 92697, USA
- Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697, USA
- Department of Anatomy and Neurobiology, School of Medicine, University of California Irvine, Irvine, CA 92697, USA
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49
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Bae J, Kitayama S, Herbert Z, Daheron L, Kurata K, Keskin DB, Livak K, Li S, Tarannum M, Romee R, Samur M, Munshi NC, Kaneko S, Ritz J, Anderson KC. Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αβ+ T cells targeting multiple myeloma. Blood 2024; 143:895-911. [PMID: 37890146 PMCID: PMC10940063 DOI: 10.1182/blood.2023020528] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 10/16/2023] [Accepted: 10/17/2023] [Indexed: 10/29/2023] Open
Abstract
ABSTRACT A major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14- CD235a-), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αβ+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC-T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αβ+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αβ+ memory T cells to improve the outcome in patients with MM.
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Affiliation(s)
- Jooeun Bae
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Shuichi Kitayama
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Zach Herbert
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | | | - Keiji Kurata
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Derin B. Keskin
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Kenneth Livak
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Shuqiang Li
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
| | - Mubin Tarannum
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Rizwan Romee
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Mehmet Samur
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Nikhil C. Munshi
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Shin Kaneko
- Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Jerome Ritz
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
| | - Kenneth C. Anderson
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
- Harvard Medical School, Boston, MA
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50
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Alowaysi M, Al-Shehri M, Badkok A, Attas H, Aboalola D, Baadhaim M, Alzahrani H, Daghestani M, Zia A, Al-Ghamdi K, Al-Ghamdi A, Zakri S, Aouabdi S, Tegner J, Alsayegh K. Generation of iPSC lines (KAIMRCi003A, KAIMRCi003B) from a Saudi patient with Dravet syndrome carrying homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A. Hum Cell 2024; 37:502-510. [PMID: 38110787 PMCID: PMC10890977 DOI: 10.1007/s13577-023-01016-z] [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: 09/26/2023] [Accepted: 11/15/2023] [Indexed: 12/20/2023]
Abstract
The most prevalent form of epileptic encephalopathy is Dravet syndrome (DRVT), which is triggered by the pathogenic variant SCN1A in 80% of cases. iPSCs with different SCN1A mutations have been constructed by several groups to model DRVT syndrome. However, no studies involving DRVT-iPSCs with rare genetic variants have been conducted. Here, we established two DRVT-iPSC lines harboring a homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A gene. Therefore, the derivation of these iPSC lines provides a unique cellular platform to dissect the molecular mechanisms underlying the cellular dysfunctions consequent to CPLX1 and SCN9A mutations.
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Affiliation(s)
- Maryam Alowaysi
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Mohammad Al-Shehri
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Amani Badkok
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Hanouf Attas
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Doaa Aboalola
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Moayad Baadhaim
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Hajar Alzahrani
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Mustafa Daghestani
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
- Department of Pathology and Laboratory Medicine, Ministry of the National Guard-Health Affairs, Jeddah, Saudi Arabia
| | - Asima Zia
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
| | - Khalid Al-Ghamdi
- Forensic Laboratories, Criminal Evidence Department, Jeddah, Saudi Arabia
| | - Asayil Al-Ghamdi
- Forensic Laboratories, Criminal Evidence Department, Jeddah, Saudi Arabia
| | - Samer Zakri
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Sihem Aouabdi
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Jesper Tegner
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
- Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
| | - Khaled Alsayegh
- King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia.
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