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Li ZK, Wang LB, Wang LY, Sun XH, Ren ZH, Ma SN, Zhao YL, Liu C, Feng GH, Liu T, Pan TS, Shan QT, Xu K, Luo GZ, Zhou Q, Li W. Adult bi-paternal offspring generated through direct modification of imprinted genes in mammals. Cell Stem Cell 2025; 32:361-374.e6. [PMID: 39879989 DOI: 10.1016/j.stem.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: 11/09/2024] [Revised: 12/24/2024] [Accepted: 01/06/2025] [Indexed: 01/31/2025]
Abstract
Imprinting abnormalities pose a significant challenge in applications involving embryonic stem cells, induced pluripotent stem cells, and animal cloning, with no universal correction method owing to their complexity and stochastic nature. In this study, we targeted these defects at their source-embryos from same-sex parents-aiming to establish a stable, maintainable imprinting pattern de novo in mammalian cells. Using bi-paternal mouse embryos, which exhibit severe imprinting defects and are typically non-viable, we introduced frameshift mutations, gene deletions, and regulatory edits at 20 key imprinted loci, ultimately achieving the development of fully adult animals, albeit with a relatively low survival rate. The findings provide strong evidence that imprinting abnormalities are a primary barrier to unisexual reproduction in mammals. Moreover, this approach can significantly improve developmental outcomes for embryonic stem cells and cloned animals, opening promising avenues for advancements in regenerative medicine.
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Affiliation(s)
- Zhi-Kun Li
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Li-Bin Wang
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Le-Yun Wang
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Xue-Han Sun
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ze-Hui Ren
- MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
| | - Si-Nan Ma
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Yu-Long Zhao
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chao Liu
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Gui-Hai Feng
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Tao Liu
- Beijing SeqWisdom Biotechnology Co., Ltd., Beijing 100176, China
| | - Tian-Shi Pan
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Qing-Tong Shan
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Kai Xu
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Guan-Zheng Luo
- MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
| | - Qi Zhou
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Wei Li
- State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
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Ishida-Ishihara S, Yaguchi K, Miura S, Nomura R, Wang Q, Yoshizawa K, Sato K, Yang G, Veszelyi K, Banhegyi G, Margittai E, Uehara R. Fragility of ER homeostatic regulation underlies haploid instability in human somatic cells. J Biol Chem 2024; 300:107909. [PMID: 39433129 DOI: 10.1016/j.jbc.2024.107909] [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: 04/06/2024] [Revised: 09/29/2024] [Accepted: 10/08/2024] [Indexed: 10/23/2024] Open
Abstract
Mammalian somatic cells are generally unstable in the haploid state, resulting in haploid-to-diploid conversion within a short time frame. However, cellular and molecular principles that limit the sustainability of somatic haploidy remain unknown. In this study, we found the haploidy-linked vulnerability to endoplasmic reticulum (ER) stress as a critical cause of haploid intolerance in human somatic cells. Pharmacological induction of ER stress selectively induced apoptosis in haploid cells, facilitating the replacement of haploids by coexisting diploidized cells in a caspase-dependent manner. Biochemical analyses revealed that unfolded protein response (UPR) was activated with similar dynamics between haploids and diploids upon ER stress induction. However, haploids were less efficient in solving proteotoxic stress, resulting in a bias toward a proapoptotic mode of UPR signaling. Artificial replenishment of chaperone function substantially alleviated the haploidy-linked upregulation of proapoptotic signaling and improved haploid cell retention under tunicamycin-induced ER stress. These data demonstrate that the ER stress-driven haploid instability stems from inefficient proteostatic control that alters the functionality of UPR to cause apoptosis selectively in haploids. Interestingly, haploids suffered a higher level of protein aggregation even in unperturbed conditions, and the long-term stability of the haploid state was significantly improved by alleviating their natural proteotoxicity. Based on these results, we propose that the haploidy-specific vulnerability to ER stress creates a fundamental cause of haploid intolerance in mammalian somatic cells. Our findings provide new insight into the principle that places a stringent restriction on the evolution of animal life cycles.
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Affiliation(s)
- Sumire Ishida-Ishihara
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
| | - Kan Yaguchi
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
| | - Sena Miura
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Ryoto Nomura
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - QiJiao Wang
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Koya Yoshizawa
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Kimino Sato
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Guang Yang
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Krisztina Veszelyi
- Institute of Translational Medicine, Semmelweis University, Budapest, Hungary
| | - Gabor Banhegyi
- Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Eva Margittai
- Institute of Translational Medicine, Semmelweis University, Budapest, Hungary
| | - Ryota Uehara
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
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Wang HS, Ma XR, Guo YH. Development and application of haploid embryonic stem cells. Stem Cell Res Ther 2024; 15:116. [PMID: 38654389 PMCID: PMC11040874 DOI: 10.1186/s13287-024-03727-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Accepted: 04/10/2024] [Indexed: 04/25/2024] Open
Abstract
Haploid cells are a kind of cells with only one set of chromosomes. Compared with traditional diploid cells, haploid cells have unique advantages in gene screening and drug-targeted therapy, due to their phenotype being equal to the genotype. Embryonic stem cells are a kind of cells with strong differentiation potential that can differentiate into various types of cells under specific conditions in vitro. Therefore, haploid embryonic stem cells have the characteristics of both haploid cells and embryonic stem cells, which makes them have significant advantages in many aspects, such as reproductive developmental mechanism research, genetic screening, and drug-targeted therapy. Consequently, establishing haploid embryonic stem cell lines is of great significance. This paper reviews the progress of haploid embryonic stem cell research and briefly discusses the applications of haploid embryonic stem cells.
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Affiliation(s)
- Hai-Song Wang
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, No. 40 Daxue Road, 450052, Zhengzhou, Henan Province, China.
| | - Xin-Rui Ma
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, No. 40 Daxue Road, 450052, Zhengzhou, Henan Province, China
| | - Yi-Hong Guo
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, No. 40 Daxue Road, 450052, Zhengzhou, Henan Province, China.
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Zheng W, Wang L, He W, Hu X, Zhu Q, Gu L, Jiang C. Transcriptome profiles and chromatin states in mouse androgenetic haploid embryonic stem cells. Cell Prolif 2023; 56:e13436. [PMID: 36855927 PMCID: PMC10472531 DOI: 10.1111/cpr.13436] [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: 11/14/2022] [Revised: 02/12/2023] [Accepted: 02/17/2023] [Indexed: 03/02/2023] Open
Abstract
Haploid embryonic stem cells (haESCs) are derived from the inner cell mass of the haploid blastocyst, containing only one set of chromosomes. Extensive and accurate chromatin remodelling occurs during haESC derivation, but the intrinsic transcriptome profiles and chromatin structure of haESCs have not been fully explored. We profiled the transcriptomes, nucleosome positioning, and key histone modifications of four mouse haESC lines, and compared these profiles with those of other closely-related stem cell lines, MII oocytes, round spermatids, sperm, and mouse embryonic fibroblasts. haESCs had transcriptome profiles closer to those of naïve pluripotent stem cells. Consistent with the one X chromosome in haESCs, Xist was repressed, indicating no X chromosome inactivation. haESCs and ESCs shared a similar global chromatin structure. However, a nucleosome depletion region was identified in 2056 promoters in ESCs, which was absent in haESCs. Furthermore, three characteristic spatial relationships were formed between transcription factor motifs and nucleosomes in both haESCs and ESCs, specifically in the linker region, on the nucleosome central surface, and nucleosome borders. Furthermore, the chromatin state of 4259 enhancers was off in haESCs but active in ESCs. Functional annotation of these enhancers revealed enrichment in regulation of the cell cycle, a predominantly reported mechanism of haESC self-diploidization. Notably, the transcriptome profiles and chromatin structure of haESCs were highly preserved during passaging but different from those of differentiated cell types.
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Affiliation(s)
- Weisheng Zheng
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Liping Wang
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Wenteng He
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Xinjie Hu
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Qianshu Zhu
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Liang Gu
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Cizhong Jiang
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and TechnologyTongji UniversityShanghaiChina
- Frontier Science Center for Stem Cell ResearchTongji UniversityShanghaiChina
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Wang HS, Ma XR, Niu WB, Shi H, Liu YD, Ma NZ, Zhang N, Jiang ZW, Sun YP. Generation of a human haploid neural stem cell line for genome-wide genetic screening. World J Stem Cells 2023; 15:734-750. [PMID: 37545755 PMCID: PMC10401418 DOI: 10.4252/wjsc.v15.i7.734] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 06/01/2023] [Accepted: 06/21/2023] [Indexed: 07/25/2023] Open
Abstract
BACKGROUND Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.
AIM To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens.
METHODS Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools.
RESULTS Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs’ genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools.
CONCLUSION This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.
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Affiliation(s)
- Hai-Song Wang
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Xin-Rui Ma
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Wen-Bin Niu
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Hao Shi
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Yi-Dong Liu
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Ning-Zhao Ma
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Nan Zhang
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Zi-Wei Jiang
- Basic Medical School, Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Ying-Pu Sun
- Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
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Hamada T, Yokoyama S, Akahane T, Matsuo K, Tanimoto A. Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines. Int J Mol Sci 2022; 24:ijms24010500. [PMID: 36613947 PMCID: PMC9820287 DOI: 10.3390/ijms24010500] [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: 11/19/2022] [Revised: 12/12/2022] [Accepted: 12/21/2022] [Indexed: 12/29/2022] Open
Abstract
Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.
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Wang H, Ma X, Niu W, Shi H, Liu Y, Ma N, Zhang N, Sun Y. Generation of human haploid neural stem cells from parthenogenetic embryonic stem cells.. [DOI: 10.21203/rs.3.rs-2332761/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/29/2023]
Abstract
Abstract
Recently, haploid embryonic stem cells (haESCs) have been established in many species and widely used in forward and reverse genetic screening. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens. Human embryonic stem cells are widely used in basic and preclinical research. In this work, we report that human haESCs in extended pluripotent culture medium showed more compact colonies, higher efficiency in neural differentiation, and higher stability in haploidy maintenance, which effectively facilitated the derivation of haNSCs. Human haploid neural stem cells (haNSCs) can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the haNSC genome and the insertion sites evenly spread across all chromosomes. This is the first human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential, which provides cell resources for recessive inheritance and drug targeted screening.
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Affiliation(s)
- Haisong Wang
- The First Affiliated Hospital of Zhengzhou University
| | - Xinrui Ma
- The First Affiliated Hospital of Zhengzhou University
| | - Wenbin Niu
- The First Affiliated Hospital of Zhengzhou University
| | - Hao Shi
- The First Affiliated Hospital of Zhengzhou University
| | - Yidong Liu
- The First Affiliated Hospital of Zhengzhou University
| | - Ningzhao Ma
- The First Affiliated Hospital of Zhengzhou University
| | - Nan Zhang
- The First Affiliated Hospital of Zhengzhou University
| | - Ying-Pu Sun
- The First Affiliated Hospital of zhengzhou university
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Xu M, Zhang W, Geng M, Zhao Y, Sun S, Gao Q, Liu Y, Shuai L. Rapid generation of murine haploid-induced trophoblast stem cells via a Tet-on system. STAR Protoc 2021; 2:100881. [PMID: 34806042 PMCID: PMC8585661 DOI: 10.1016/j.xpro.2021.100881] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Haploid trophoblast stem cells (TSCs) are advanced in studying placental development for their placental precursor and homozygous features. Here, we describe how to generate haploid-induced TSCs (haiTSCs) from haploid embryonic stem cells with a Tet-on system. Our haiTSCs can maintain haploidy long-term and can produce genome-wide mutants combined with transposons. It is promising in high-throughput genetic screening of trophoblast-specific modulators. For complete details on the use and execution of this protocol, please refer to Peng et al. (2019).
Protocol for inducing a Cdx2-OE Tet-on system into haploid ESCs Protocol for conversion and purification of haploid induced TSCs from haploid ESCs Protocol for construction of genome-wide mutated homozygous TSCs by piggyBac transposon
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Affiliation(s)
- Mei Xu
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Wenhao Zhang
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Mengyang Geng
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Yiding Zhao
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Shengyi Sun
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Qian Gao
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China
| | - Yan Liu
- Department of Obstetrics, Tianjin First Central Hospital, Nankai University, Tianjin 300192, China
| | - Ling Shuai
- State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Nankai University, Tianjin 300350, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China
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9
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Applications of piggyBac Transposons for Genome Manipulation in Stem Cells. Stem Cells Int 2021; 2021:3829286. [PMID: 34567130 PMCID: PMC8460389 DOI: 10.1155/2021/3829286] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Accepted: 08/16/2021] [Indexed: 12/20/2022] Open
Abstract
Transposons are mobile genetic elements in the genome. The piggyBac (PB) transposon system is increasingly being used for stem cell research due to its high transposition efficiency and seamless excision capacity. Over the past few decades, forward genetic screens based on PB transposons have been successfully established to identify genes associated with drug resistance and stem cell-related characteristics. Moreover, PB transposon is regarded as a promising gene therapy vector and has been used in some clinically relevant stem cells. Here, we review the recent progress on the basic biology of PB, highlight its applications in current stem cell research, and discuss its advantages and challenges.
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Gao Q, Zhang W, Zhao Y, Tian Y, Wang Y, Zhang J, Geng M, Xu M, Yao C, Wang H, Li L, Liu Y, Shuai L. High-throughput screening in postimplantation haploid epiblast stem cells reveals Hs3st3b1 as a modulator for reprogramming. Stem Cells Transl Med 2021; 10:743-755. [PMID: 33511777 PMCID: PMC8046116 DOI: 10.1002/sctm.20-0468] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 12/12/2020] [Accepted: 01/03/2021] [Indexed: 12/15/2022] Open
Abstract
Epiblast stem cells (EpiSCs) derived from postimplantation epiblast are pluripotent stem cells, epigenetically distinct from embryonic stem cells (ESCs), which are widely used in reprogramming studies. Recent achieved haploid cell lines in mammalian species open a new era for high-throughput genetic screening, due to their homozygous phenotypes. Here, we report the generation of mouse haploid EpiSCs (haEpiSCs) from postimplantation chimeric embryos at embryonic day 6.5 (E6.5). These cells maintain one set of chromosomes, express EpiSC-specific genes, and have potentials to differentiate into three germ layers. We also develop a massive mutagenesis protocol with haEpiSCs, and subsequently perform reprogramming selection using this genome-wide mutation library. Multiple modules related to various pathways are implicated. The validation experiments prove that knockout of Hst3st3b1 (one of the candidates) can promote reprogramming of EpiSCs to the ground state efficiently. Our results open the feasibility of utilizing haEpiSCs to elucidate fundamental biological processes including cell fate alternations.
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Affiliation(s)
- Qian Gao
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Wenhao Zhang
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Yiding Zhao
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Yaru Tian
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Yuna Wang
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Jinxin Zhang
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Mengyang Geng
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Mei Xu
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Chunmeng Yao
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Haoyu Wang
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Luyuan Li
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China
| | - Yan Liu
- Department of Obstetrics, Tianjin First Central Hospital, Nankai University, Tianjin, People's Republic of China
| | - Ling Shuai
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China.,Nankai Animal Resource Center, Nankai University, Tianjin, People's Republic of China.,Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Tianjin, People's Republic of China
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Kumar D, Talluri TR, Selokar NL, Hyder I, Kues WA. Perspectives of pluripotent stem cells in livestock. World J Stem Cells 2021; 13:1-29. [PMID: 33584977 PMCID: PMC7859985 DOI: 10.4252/wjsc.v13.i1.1] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/28/2020] [Accepted: 11/09/2020] [Indexed: 02/06/2023] Open
Abstract
The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.
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Affiliation(s)
- Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India.
| | - Thirumala R Talluri
- Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner 334001, India
| | - Naresh L Selokar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India
| | - Iqbal Hyder
- Department of Physiology, NTR College of Veterinary Science, Gannavaram 521102, India
| | - Wilfried A Kues
- Department of Biotechnology, Friedrich-Loeffler-Institute, Federal Institute of Animal Health, Neustadt 31535, Germany
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12
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Zhang G, Li X, Sun Y, Wang X, Liu G, Huang Y. A Genetic Screen Identifies Etl4-Deficiency Capable of Stabilizing the Haploidy in Embryonic Stem Cells. Stem Cell Reports 2021; 16:29-38. [PMID: 33440180 PMCID: PMC7815943 DOI: 10.1016/j.stemcr.2020.11.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2020] [Revised: 11/25/2020] [Accepted: 11/26/2020] [Indexed: 01/05/2023] Open
Abstract
Mammalian haploid embryonic stem cells (haESCs) hold great promise for functional genetic studies and forward screening. However, all established haploid cells are prone to spontaneous diploidization during long-term culture, rendering application challenging. Here, we report a genome-wide loss-of-function screening that identified gene mutations that could significantly reduce the rate of self-diploidization in haESCs. We further demonstrated that CRISPR/Cas9-mediated Etl4 knockout (KO) stabilizes the haploid state in different haESC lines. More interestingly, Etl4 deficiency increases mitochondrial oxidative phosphorylation (OXPHOS) capacity and decreases glycolysis in haESCs. Mimicking this effect by regulating the energy metabolism with drugs decreased the rate of self-diploidization. Collectively, our study identified Etl4 as a novel haploidy-related factor linked to an energy metabolism transition occurring during self-diploidization of haESCs.
A genome-wide genetic screen identifies several haploidy-related factors in haESCs Etl4-deficiency stabilizes the haploid state in different haESC lines Etl4-deficiency increases mitochondrial OXPHOS and decrease glycolysis in haESCs Energy metabolism regulation with drugs decreased the rate of self-diploidization
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Affiliation(s)
- Guozhong Zhang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
| | - Xiaowen Li
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
| | - Yi Sun
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
| | - Xue Wang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
| | - Guang Liu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
| | - Yue Huang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
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