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Fan S, Guo C, Yang G, Hong L, Li H, Ma J, Zhou Y, Fan S, Xue Y, Zeng F. GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signaling pathway. J Genet Genomics 2024; 51:1055-1065. [PMID: 38750952 DOI: 10.1016/j.jgg.2024.05.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2024] [Revised: 05/08/2024] [Accepted: 05/09/2024] [Indexed: 07/14/2024]
Abstract
G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) remain poorly understood. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of Gpr160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Consistent with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway.
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Affiliation(s)
- Shasha Fan
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Chuanliang Guo
- Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Guanheng Yang
- Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Lei Hong
- Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Hongyu Li
- Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Ji Ma
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Yiye Zhou
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Shuyue Fan
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
| | - Yan Xue
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China.
| | - Fanyi Zeng
- Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China; School of Pharmacy, Macau University of Science and Technology, Macau 999078, China.
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Ealo T, Sanchez-Gaya V, Respuela P, Muñoz-San Martín M, Martin-Batista E, Haro E, Rada-Iglesias A. Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition. Nat Commun 2024; 15:7258. [PMID: 39179577 PMCID: PMC11344162 DOI: 10.1038/s41467-024-51602-4] [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: 01/23/2024] [Accepted: 08/10/2024] [Indexed: 08/26/2024] Open
Abstract
The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.
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Affiliation(s)
- Thais Ealo
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain
| | - Victor Sanchez-Gaya
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain.
| | - Patricia Respuela
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain
| | - María Muñoz-San Martín
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain
- Service of Neurology, University Hospital Marqués de Valdecilla, Universidad de Cantabria and IDIVAL, Santander, Spain
| | | | - Endika Haro
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain.
| | - Alvaro Rada-Iglesias
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Santander, Spain.
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Wei Q, Xu Y, Cui G, Sun J, Su Z, Kou X, Zhao Y, Cao S, Li W, Xu Y, Gao S. Male-pronuclei-specific granulin facilitates somatic cells reprogramming via mitigating excessive cell proliferation and enhancing lysosomal function. J Cell Physiol 2024; 239:e31295. [PMID: 38747637 DOI: 10.1002/jcp.31295] [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: 11/01/2023] [Revised: 04/05/2024] [Accepted: 04/30/2024] [Indexed: 08/15/2024]
Abstract
Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome-related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.
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Affiliation(s)
- Qingqing Wei
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Yanwen Xu
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Guina Cui
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Jiatong Sun
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Zhongqu Su
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Xiaochen Kou
- Clinical and Translation Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China
| | - Yanhong Zhao
- Clinical and Translation Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China
| | - Suyuan Cao
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Wenhui Li
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Yiliang Xu
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China
| | - Shaorong Gao
- Clinical and Translation Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China
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Tan H, Miao MX, Luo RX, So J, Peng L, Zhu X, Leung EHW, Zhu L, Chan KM, Cheung M, Chan SY. TSPYL1 as a Critical Regulator of TGFβ Signaling through Repression of TGFBR1 and TSPYL2. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2306486. [PMID: 38588050 PMCID: PMC11151076 DOI: 10.1002/advs.202306486] [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] [Subscribe] [Scholar Register] [Received: 09/08/2023] [Revised: 02/20/2024] [Indexed: 04/10/2024]
Abstract
Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFβ causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFβ signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFβ signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFβ to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFβ signaling is also unraveled.
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Affiliation(s)
- Huiqi Tan
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Mia Xinfang Miao
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Rylee Xu Luo
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Joan So
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Lei Peng
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Xiaoxuan Zhu
- Department of Biomedical Sciences, The City University of Hong Kong, Hong Kong, China
| | - Eva Hin Wa Leung
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Lina Zhu
- Department of Biomedical Sciences, The City University of Hong Kong, Hong Kong, China
| | - Kui Ming Chan
- Department of Biomedical Sciences, The City University of Hong Kong, Hong Kong, China
| | - Martin Cheung
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Siu Yuen Chan
- Department of Paediatrics and Adolescent Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
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Bekas N, Samiotaki M, Papathanasiou M, Mokos P, Pseftogas A, Xanthopoulos K, Thanos D, Mosialos G, Dafou D. Inactivation of Tumor Suppressor CYLD Inhibits Fibroblast Reprogramming to Pluripotency. Cancers (Basel) 2023; 15:4997. [PMID: 37894364 PMCID: PMC10605754 DOI: 10.3390/cancers15204997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2023] [Accepted: 10/12/2023] [Indexed: 10/29/2023] Open
Abstract
CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLDΔ9/Δ9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLDΔ9/Δ9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.
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Affiliation(s)
- Nikolaos Bekas
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Martina Samiotaki
- Biomedical Sciences Research Center “Alexander Fleming”, 16672 Vari, Greece;
| | - Maria Papathanasiou
- Biomedical Research Foundation Academy of Athens, 11527 Athens, Greece; (M.P.); (D.T.)
| | - Panagiotis Mokos
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Athanasios Pseftogas
- Division of Experimental Oncology, IRCCS San Raffaele Hospital, Vita-Salute San Raffaele University, 20132 Milan, Italy;
| | - Konstantinos Xanthopoulos
- Laboratory of Pharmacology, Department of Pharmacy, School of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Dimitris Thanos
- Biomedical Research Foundation Academy of Athens, 11527 Athens, Greece; (M.P.); (D.T.)
| | - George Mosialos
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Dimitra Dafou
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
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Li X, Chen P, Ji J, Duan Q, Cao J, Huang R, Ye SD. Rhox6 regulates the expression of distinct target genes to mediate mouse PGCLC formation and ESC self-renewal. Cell Biosci 2023; 13:145. [PMID: 37553721 PMCID: PMC10408072 DOI: 10.1186/s13578-023-01096-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Accepted: 07/30/2023] [Indexed: 08/10/2023] Open
Abstract
BACKGROUND Mouse embryonic stem cells (mESCs) not only retain the property of self-renewal but also have the ability to develop into primordial germ cell-like cells (PGCLCs). However, knowledge about the mechanisms of transcriptional regulation is still limited. Rhox6, a member of the homeobox family that is located on the X chromosome, is highly expressed within PGCLCs in vivo and in vitro. However, the detailed effects of Rhox6 on PGCLC specification and mESC maintenance remain unclear. RESULTS In this study, we found that overexpression of Rhox6 favors the formation of PGCLCs, while depletion of Rhox6 inhibits the generation of PGCLCs. Mechanistically, Rhox6 directly induces the expression of Nanos3 during the specification of PGCLCs. Subsequently, downregulation of Nanos3 expression is sufficient to decrease the ability of Rhox6 to induce PGCLC formation. Moreover, we found that depletion of Rhox6 expression facilitates the self-renewal of mESCs. High-throughput sequencing revealed that suppression of Rhox6 transcription significantly increases the expression of pluripotency genes. Functional studies further demonstrated that Rhox6 directly represses the transcription of Tbx3. Therefore, knockdown of the expression of the latter impairs the self-renewal of mESCs promoted by Rhox6 downregulation. CONCLUSIONS Our study reveals that overexpression of Rhox6 is beneficial for PGCLC generation through induction of Nanos3, while downregulation of Rhox6 contributes to mESC self-renewal by increasing Tbx3. These findings help elucidate the early development of mouse embryos.
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Affiliation(s)
- Xiaofeng Li
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Peng Chen
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Junxiang Ji
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Quanchao Duan
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Jianjian Cao
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Ru Huang
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China
| | - Shou-Dong Ye
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China.
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Miyazaki S, Yamano H, Motooka D, Tashiro F, Matsuura T, Miyazaki T, Miyazaki JI. Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells. Commun Biol 2023; 6:771. [PMID: 37488353 PMCID: PMC10366109 DOI: 10.1038/s42003-023-05148-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Accepted: 07/17/2023] [Indexed: 07/26/2023] Open
Abstract
The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.
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Affiliation(s)
- Satsuki Miyazaki
- Division of Stem Cell Regulation Research, Center for Medical Research and Education, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Hiroyuki Yamano
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Daisuke Motooka
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Fumi Tashiro
- The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan
| | - Takumi Matsuura
- Division of Stem Cell Regulation Research, Center for Medical Research and Education, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
- Toray Industries, Inc., Tokyo, Japan
| | - Tatsushi Miyazaki
- Division of Stem Cell Regulation Research, Center for Medical Research and Education, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Jun-Ichi Miyazaki
- The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.
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Correia B, Sousa MI, Branco AF, Rodrigues AS, Ramalho-Santos J. Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism. Int J Mol Sci 2022; 23:ijms232214286. [PMID: 36430764 PMCID: PMC9694364 DOI: 10.3390/ijms232214286] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 11/08/2022] [Accepted: 11/09/2022] [Indexed: 11/19/2022] Open
Abstract
Amino acids are crucial nutrients involved in several cellular and physiological processes, including fertilization and early embryo development. In particular, Leucine and Arginine have been shown to stimulate implantation, as lack of both in a blastocyst culture system is able to induce a dormant state in embryos. The aim of this work was to evaluate the effects of Leucine and Arginine withdrawal on pluripotent mouse embryonic stem cell status, notably, their growth, self-renewal, as well as glycolytic and oxidative metabolism. Our results show that the absence of both Leucine and Arginine does not affect mouse embryonic stem cell pluripotency, while reducing cell proliferation through cell-cycle arrest. Importantly, these effects are not related to Leukemia Inhibitory Factor (LIF) and are reversible when both amino acids are reconstituted in the culture media. Moreover, a lack of these amino acids is related to a reduction in glycolytic and oxidative metabolism and decreased protein translation in mouse embryonic stem cells (mESCs), while maintaining their pluripotent status.
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Affiliation(s)
- Bibiana Correia
- Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
- CNC—Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Polo 3, 3000-354 Coimbra, Portugal
| | - Maria Inês Sousa
- Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
- CNC—Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Polo 3, 3000-354 Coimbra, Portugal
| | - Ana Filipa Branco
- CNC—Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Polo 3, 3000-354 Coimbra, Portugal
| | - Ana Sofia Rodrigues
- CNC—Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Polo 3, 3000-354 Coimbra, Portugal
| | - João Ramalho-Santos
- Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
- CNC—Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Polo 3, 3000-354 Coimbra, Portugal
- Correspondence:
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9
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Pladevall-Morera D, Zylicz JJ. Chromatin as a sensor of metabolic changes during early development. Front Cell Dev Biol 2022; 10:1014498. [PMID: 36299478 PMCID: PMC9588933 DOI: 10.3389/fcell.2022.1014498] [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: 08/08/2022] [Accepted: 09/13/2022] [Indexed: 11/13/2022] Open
Abstract
Cellular metabolism is a complex network of biochemical reactions fueling development with energy and biomass; however, it can also shape the cellular epigenome. Indeed, some intermediates of metabolic reactions exert a non-canonical function by acting as co-factors, substrates or inhibitors of chromatin modifying enzymes. Therefore, fluctuating availability of such molecules has the potential to regulate the epigenetic landscape. Thanks to this functional coupling, chromatin can act as a sensor of metabolic changes and thus impact cell fate. Growing evidence suggest that both metabolic and epigenetic reprogramming are crucial for ensuring a successful embryo development from the zygote until gastrulation. In this review, we provide an overview of the complex relationship between metabolism and epigenetics in regulating the early stages of mammalian embryo development. We report on recent breakthroughs in uncovering the non-canonical functions of metabolism especially when re-localized to the nucleus. In addition, we identify the challenges and outline future perspectives to advance the novel field of epi-metabolomics especially in the context of early development.
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Affiliation(s)
| | - Jan J. Zylicz
- Novo Nordisk Foundation Center for Stem Cell Medicine, reNEW, University of Copenhagen, Copenhagen, Denmark
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10
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Li Y, Yang Z, Li X, Yu Y, Li X, Chen P, Li B, Wang X, Ye SD. Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity. iScience 2022; 25:105293. [PMID: 36300005 PMCID: PMC9589213 DOI: 10.1016/j.isci.2022.105293] [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: 10/26/2021] [Revised: 07/13/2022] [Accepted: 10/03/2022] [Indexed: 11/30/2022] Open
Abstract
Prdm14 plays an important role in the maintenance of mouse embryonic stem cell (mESC) pluripotency and the specification of primordial germ cells (PGCs). However, the mechanism downstream of Prdm14 is still not fully understood. Here, using high-throughput sequencing, chromatin immunoprecipitation, and luciferase reporter assays, we show that Prdm14 directly binds to the promoter of Socs3 and represses its transcription to increase the phosphorylation level of Stat3 protein, a critical downstream effector of LIF. Therefore, ectopic expression of Socs3 is able to decrease the ability of Prdm14 to promote mouse mESC self-renewal and PGC-like cell generation. As expected, similar phenotypes were observed in Prdm14-transfected mESCs after knockdown of Stat3 transcripts or treatment with a pan-inhibitor of JAKs, positive modulators of the LIF/Stat3 signaling pathway. These data will facilitate a better understanding of the regulatory network governing ESC identity and germ cell development.
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Affiliation(s)
- Yuting Li
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Ziqiong Yang
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Xiangfen Li
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Yang Yu
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Xiaofeng Li
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Peng Chen
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Bing Li
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
| | - Xiaoxiao Wang
- Core Facility Center, The First Affiliated Hospital of USTC (Anhui Provincial Hospital), Hefei, Anhui 230001, China
- Corresponding author
| | - Shou-Dong Ye
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
- Corresponding author
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11
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Cancer cells as a new source of induced pluripotent stem cells. Stem Cell Res Ther 2022; 13:459. [PMID: 36064437 PMCID: PMC9446809 DOI: 10.1186/s13287-022-03145-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 08/17/2022] [Indexed: 11/10/2022] Open
Abstract
Over the last 2 decades, induced pluripotent stem cells (iPSCs) have had various potential applications in various medical research areas, from personalized medicine to disease treatment. Different cellular resources are accessible for iPSC generation, such as keratinocytes, skin fibroblasts, and blood or urine cells. However, all these sources are somatic cells, and we must make several changes in a somatic cell's transcriptome and chromatin state to become a pluripotent cell. It has recently been revealed that cancer cells can be a new source of iPSCs production. Cancer cells show similarities with iPSCs in self-renewal capacity, reprogramming potency, and signaling pathways. Although genetic abnormalities and potential tumor formation in cancer cells pose a severe risk, reprogrammed cancer-induced pluripotent stem cells (cancer-iPSCs) indicate that pluripotency can transiently overcome the cancer phenotype. This review discusses whether cancer cells can be a preferable source to generate iPSCs.
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12
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Roodgar M, Suchy FP, Nguyen LH, Bajpai VK, Sinha R, Vilches-Moure JG, Van Bortle K, Bhadury J, Metwally A, Jiang L, Jian R, Chiang R, Oikonomopoulos A, Wu JC, Weissman IL, Mankowski JL, Holmes S, Loh KM, Nakauchi H, VandeVoort CA, Snyder MP. Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos. Cell Rep 2022; 40:111264. [PMID: 36044843 PMCID: PMC10075238 DOI: 10.1016/j.celrep.2022.111264] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2020] [Revised: 01/06/2022] [Accepted: 08/04/2022] [Indexed: 12/26/2022] Open
Abstract
As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.
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Affiliation(s)
- Morteza Roodgar
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
| | - Fabian P Suchy
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Lan H Nguyen
- Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA 94305, USA
| | - Vivek K Bajpai
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
| | - Rahul Sinha
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Jose G Vilches-Moure
- Department of Comparative Medicine, Stanford University, Stanford, CA 94305, USA
| | - Kevin Van Bortle
- Department of Genetics, Stanford University, Stanford, CA 94305, USA
| | - Joydeep Bhadury
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, SE 413 45 Gothenburg, Sweden
| | - Ahmed Metwally
- Department of Genetics, Stanford University, Stanford, CA 94305, USA
| | - Lihua Jiang
- Department of Genetics, Stanford University, Stanford, CA 94305, USA
| | - Ruiqi Jian
- Department of Genetics, Stanford University, Stanford, CA 94305, USA
| | - Rosaria Chiang
- Department of Genetics, Stanford University, Stanford, CA 94305, USA
| | - Angelos Oikonomopoulos
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Joseph C Wu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Irving L Weissman
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Joseph L Mankowski
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Susan Holmes
- Department of Statistics, Stanford University, Stanford, CA 94305, USA
| | - Kyle M Loh
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Hiromitsu Nakauchi
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
| | - Catherine A VandeVoort
- California National Primate Research Center and Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA, USA.
| | - Michael P Snyder
- Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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13
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Andersson E, Sjö M, Kaji K, Olariu V. CELLoGeNe - An energy landscape framework for logical networks controlling cell decisions. iScience 2022; 25:104743. [PMID: 35942105 PMCID: PMC9356104 DOI: 10.1016/j.isci.2022.104743] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Revised: 06/01/2022] [Accepted: 07/05/2022] [Indexed: 11/29/2022] Open
Abstract
Experimental and computational efforts are constantly made to elucidate mechanisms controlling cell fate decisions during development and reprogramming. One powerful computational method is to consider cell commitment and reprogramming as movements in an energy landscape. Here, we develop Computation of Energy Landscapes of Logical Gene Networks (CELLoGeNe), which maps Boolean implementation of gene regulatory networks (GRNs) into energy landscapes. CELLoGeNe removes inadvertent symmetries in the energy landscapes normally arising from standard Boolean operators. Furthermore, CELLoGeNe provides tools to visualize and stochastically analyze the shapes of multi-dimensional energy landscapes corresponding to epigenetic landscapes for development and reprogramming. We demonstrate CELLoGeNe on two GRNs governing different aspects of induced pluripotent stem cells, identifying experimentally validated attractors and revealing potential reprogramming roadblocks. CELLoGeNe is a general framework that can be applied to various biological systems offering a broad picture of intracellular dynamics otherwise inaccessible with existing methods.
CELLoGeNe – Computation of Energy Landscapes of Logical Gene Networks Cell states as landscape attractors Maintenance and acquisition of cell pluripotency applications Single cell stochastic landscape navigation and visualization tool
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14
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Mustafi P, Hu M, Kumari S, Das C, Li G, Kundu T. Phosphorylation-dependent association of human chromatin protein PC4 to linker histone H1 regulates genome organization and transcription. Nucleic Acids Res 2022; 50:6116-6136. [PMID: 35670677 PMCID: PMC9226532 DOI: 10.1093/nar/gkac450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 05/08/2022] [Accepted: 05/30/2022] [Indexed: 11/16/2022] Open
Abstract
Human Positive Coactivator 4 (PC4) is a multifaceted chromatin protein involved in diverse cellular processes including genome organization, transcription regulation, replication, DNA repair and autophagy. PC4 exists as a phospho-protein in cells which impinges on its acetylation by p300 and thereby affects its transcriptional co-activator functions via double-stranded DNA binding. Despite the inhibitory effects, the abundance of phosphorylated PC4 in cells intrigued us to investigate its role in chromatin functions in a basal state of the cell. We found that casein kinase-II (CKII)-mediated phosphorylation of PC4 is critical for its interaction with linker histone H1. By employing analytical ultracentrifugation and electron microscopy imaging of in vitro reconstituted nucleosomal array, we observed that phospho-mimic (PM) PC4 displays a superior chromatin condensation potential in conjunction with linker histone H1. ATAC-sequencing further unveiled the role of PC4 phosphorylation to be critical in inducing chromatin compaction of a wide array of coding and non-coding genes in vivo. Concordantly, phospho-PC4 mediated changes in chromatin accessibility led to gene repression and affected global histone modifications. We propose that the abundance of PC4 in its phosphorylated state contributes to genome compaction contrary to its co-activator function in driving several cellular processes like gene transcription and autophagy.
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Affiliation(s)
- Pallabi Mustafi
- Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
| | - Mingli Hu
- National laboratory of Bio-macromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China
| | - Sujata Kumari
- Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
| | - Chandrima Das
- Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India
| | - Guohong Li
- National laboratory of Bio-macromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China
| | - Tapas K Kundu
- Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
- Division of Neuroscience and Ageing Biology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10, Jankipuram Extension, Lucknow 226031, India
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15
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BMP4 drives primed to naïve transition through PGC-like state. Nat Commun 2022; 13:2756. [PMID: 35589713 PMCID: PMC9120449 DOI: 10.1038/s41467-022-30325-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Accepted: 04/26/2022] [Indexed: 11/09/2022] Open
Abstract
Multiple pluripotent states have been described in mouse and human stem cells. Here, we apply single-cell RNA-seq to a newly established BMP4 induced mouse primed to naïve transition (BiPNT) system and show that the reset is not a direct reversal of cell fate but goes through a primordial germ cell-like cells (PGCLCs) state. We first show that epiblast stem cells bifurcate into c-Kit+ naïve and c-Kit- trophoblast-like cells, among which, the naïve branch undergoes further transition through a PGCLCs intermediate capable of spermatogenesis in vivo. Mechanistically, we show that DOT1L inhibition permits the transition from primed pluripotency to PGCLCs in part by facilitating the loss of H3K79me2 from Gata3/6. In addition, Prdm1/Blimp1 is required for PGCLCs and naïve cells, while Gata2 inhibits PGC-like state by promoting trophoblast-like fate. Our work not only reveals an alternative route for primed to naïve transition, but also gains insight into germ cell development.
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16
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Jamwal S, Ansari S, Malakar D, Kaushik JK, Kumar S, Mohanty AK. Production of biologically active recombinant buffalo leukemia inhibitory factor (BuLIF) in Escherichia Coli. J Genet Eng Biotechnol 2022; 20:47. [PMID: 35294648 PMCID: PMC8927517 DOI: 10.1186/s43141-022-00328-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2021] [Accepted: 02/11/2022] [Indexed: 12/27/2022]
Abstract
Background Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). Results The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. Conclusions The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-022-00328-1.
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Affiliation(s)
- Shradha Jamwal
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Shama Ansari
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Dhruba Malakar
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Jai Kumar Kaushik
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Sudarshan Kumar
- Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India.
| | - Ashok Kumar Mohanty
- Indian Council of Agricultural Research-Indian Veterinary Research Institute, Mukteshwar, India.
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17
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Wei M, Chen Y, Zhao C, Zheng L, Wu B, Chen C, Li X, Bao S. Establishment of Mouse Primed Stem Cells by Combination of Activin and LIF Signaling. Front Cell Dev Biol 2021; 9:713503. [PMID: 34422831 PMCID: PMC8375391 DOI: 10.3389/fcell.2021.713503] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Accepted: 07/09/2021] [Indexed: 01/09/2023] Open
Abstract
In mice, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are established from pre- and post-implantation embryos and represent the naive and primed state, respectively. Herein we used mouse leukemia inhibitory factor (LIF), which supports ESCs self-renewal and Activin A (Act A), which is the main factor in maintaining EpiSCs in post-implantation epiblast cultures, to derive a primed stem cell line named ALSCs. Like EpiSCs, ALSCs express key pluripotent genes Oct4, Sox2, and Nanog; one X chromosome was inactivated; and the cells failed to contribute to chimera formation in vivo. Notably, compared to EpiSCs, ALSCs efficiently reversed to ESCs (rESCs) on activation of Wnt signaling. Moreover, we also discovered that culturing EpiSCs in AL medium for several passages favored Wnt signaling-driven naive pluripotency. Our results show that ALSCs is a primed state stem cell and represents a simple model to study the control of pluripotency fate and conversion from the primed to the naive state.
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Affiliation(s)
- Mengyi Wei
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Yanglin Chen
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.,School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Chaoyue Zhao
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Li Zheng
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Baojiang Wu
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Chen Chen
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Xihe Li
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.,Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China
| | - Siqin Bao
- State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China.,Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China
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18
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Liu Y, Yamane J, Tanaka A, Fujibuchi W, Yamashita JK. AMPK activation reverts mouse epiblast stem cells to naive state. iScience 2021; 24:102783. [PMID: 34308289 PMCID: PMC8283141 DOI: 10.1016/j.isci.2021.102783] [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: 09/09/2020] [Revised: 05/01/2021] [Accepted: 06/23/2021] [Indexed: 12/25/2022] Open
Abstract
Despite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency.
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Affiliation(s)
- Yajing Liu
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Junko Yamane
- The Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Akito Tanaka
- The Department of Animal Research Facility, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Wataru Fujibuchi
- The Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Jun K. Yamashita
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
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19
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Kang X, Li C. A Dimension Reduction Approach for Energy Landscape: Identifying Intermediate States in Metabolism-EMT Network. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2003133. [PMID: 34026435 PMCID: PMC8132071 DOI: 10.1002/advs.202003133] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Revised: 11/18/2020] [Indexed: 05/08/2023]
Abstract
Dimension reduction is a challenging problem in complex dynamical systems. Here, a dimension reduction approach of landscape (DRL) for complex dynamical systems is proposed, by mapping a high-dimensional system on a low-dimensional energy landscape. The DRL approach is applied to three biological networks, which validates that new reduced dimensions preserve the major information of stability and transition of original high-dimensional systems. The consistency of barrier heights calculated from the low-dimensional landscape and transition actions calculated from the high-dimensional system further shows that the landscape after dimension reduction can quantify the global stability of the system. The epithelial-mesenchymal transition (EMT) and abnormal metabolism are two hallmarks of cancer. With the DRL approach, a quadrastable landscape for metabolism-EMT network is identified, including epithelial (E), abnormal metabolic (A), hybrid E/M (H), and mesenchymal (M) cell states. The quantified energy landscape and kinetic transition paths suggest that for the EMT process, the cells at E state need to first change their metabolism, then enter the M state. The work proposes a general framework for the dimension reduction of a stochastic dynamical system, and advances the mechanistic understanding of the underlying relationship between EMT and cellular metabolism.
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Affiliation(s)
- Xin Kang
- School of Mathematical SciencesFudan UniversityShanghai200433China
- Shanghai Center for Mathematical SciencesFudan UniversityShanghai200433China
| | - Chunhe Li
- Shanghai Center for Mathematical SciencesFudan UniversityShanghai200433China
- Institute of Science and Technology for Brain‐Inspired IntelligenceFudan UniversityShanghai200433China
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20
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Singh G, Mullany S, Moorthy SD, Zhang R, Mehdi T, Tian R, Duncan AG, Moses AM, Mitchell JA. A flexible repertoire of transcription factor binding sites and a diversity threshold determines enhancer activity in embryonic stem cells. Genome Res 2021; 31:564-575. [PMID: 33712417 PMCID: PMC8015845 DOI: 10.1101/gr.272468.120] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Accepted: 02/19/2021] [Indexed: 12/28/2022]
Abstract
Transcriptional enhancers are critical for development and phenotype evolution and are often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. To examine the enhancer regulatory code for pluripotent stem cells, we identified genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs). Examination of these regions revealed that they contain on average 12.6 conserved transcription factor binding site (TFBS) sequences. Enriched TFBSs are a diverse repertoire of 70 different sequences representing the binding sequences of both known and novel ESC regulators. Using a diverse set of TFBSs from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site-directed mutagenesis of conserved TFBSs in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for 10 or more different TFBSs. Furthermore, specific TFBSs, including the POU5F1:SOX2 comotif, are dispensable, despite cobinding the POU5F1 (also known as OCT4), SOX2, and NANOG master regulators of pluripotency. These findings reveal that a TFBS sequence diversity threshold overrides the need for optimized regulatory grammar and individual TFBSs that recruit specific master regulators.
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Affiliation(s)
- Gurdeep Singh
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Shanelle Mullany
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Sakthi D Moorthy
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Richard Zhang
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Tahmid Mehdi
- Department of Computer Science, University of Toronto, Toronto, M5S 2E4, Canada
| | - Ruxiao Tian
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Andrew G Duncan
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
| | - Alan M Moses
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada.,Department of Computer Science, University of Toronto, Toronto, M5S 2E4, Canada.,Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, M5S 3B3, Canada
| | - Jennifer A Mitchell
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada
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21
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Pluripotency of Dental Pulp Cells and Periodontal Ligament Cells Was Enhanced through Cell-Cell Communication via STAT3/Oct-4/Sox2 Signaling. Stem Cells Int 2021; 2021:8898506. [PMID: 33542738 PMCID: PMC7840254 DOI: 10.1155/2021/8898506] [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: 07/07/2020] [Revised: 12/23/2020] [Accepted: 01/02/2021] [Indexed: 02/06/2023] Open
Abstract
Alternation in culture environment due to cell-cell communications can rejuvenate the biological activity of aged/differentiated cells and stimulate the expression of pluripotency markers. Dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) are promising candidates in dental tissue regeneration. However, the molecular network that underlies cell-cell communications between dental-derived cells and the microenvironment remains to be identified. To elucidate the signaling network that regulates the pluripotency of DPCs and PDLCs, proliferation, apoptosis, cell cycle, and the expression of Oct-4/Sox2/c-Myc in DPCs and PDLCs with indirect/direct coculture were examined. PCR arrays were constructed to identify genes that were differentially expressed, and the results were confirmed by a rat model with injury. Further research on the mechanism of the related signaling pathways was investigated by overexpression/silence of STAT3, ChIP, the dual-luciferase reporter assay, and EMSA. We found that the proliferation and apoptosis of DPCs and PDLCs were inhibited, and their cell cycles were arrested at the G0/G1 phase after coculture. Oct-4, Sox2, and STAT3 expression significantly increased and PAX5 expression decreased in the coculture systems. Oct-4/Sox2/STAT3/PAX5 was actively expressed in the rat defect model. Moreover, STAT3 was directly bound to the Oct-4 and Sox2 gene promoter regions and activated the expression of those genes. Our data showed that the pluripotency of DPCs and PDLCs was enhanced through cell-cell communication. STAT3 plays essential roles in regulating the pluripotency of DPCs and PDLCs by targeting Oct-4 and Sox2 both in vitro and in vivo.
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22
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Şişli HB, Şenkal S, Sağraç D, Hayal TB, Doğan A. Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol. Methods Mol Biol 2021; 2520:101-115. [PMID: 33945144 DOI: 10.1007/7651_2021_402] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Mouse embryonic stem cells (mESCs) were first derived and cultured nearly 30 years ago and have been beneficial tools to create transgenic mice and to study early mammalian development so far. Fibroblast feeder cell layers are often used at some stage in the culture protocol of mESCs. The feeder layer-often mouse embryonic fibroblasts (MEFs)-contribute to the mESC culture as a substrate to increase culture efficiency, maintain pluripotency, and facilitate survival and growth of the stem cells. Various feeder-dependent and feeder-independent culture and differentiation protocols have been established for mESCs. Here we describe the isolation, culture, and preparation feeder cell layers and establishment of feeder-dependent/independent protocol for mESC culture. In addition, basic mESC protocols for culture, storage, and differentiation were described.
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Affiliation(s)
- Hatice Burcu Şişli
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Selinay Şenkal
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Derya Sağraç
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Taha Bartu Hayal
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
| | - Ayşegül Doğan
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey.
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23
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Swaidan NT, Salloum-Asfar S, Palangi F, Errafii K, Soliman NH, Aboughalia AT, Wali AHS, Abdulla SA, Emara MM. Identification of potential transcription factors that enhance human iPSC generation. Sci Rep 2020; 10:21950. [PMID: 33319795 PMCID: PMC7738555 DOI: 10.1038/s41598-020-78932-9] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Accepted: 11/25/2020] [Indexed: 11/09/2022] Open
Abstract
Although many factors have been identified and used to enhance the iPSC reprogramming process, its efficiency remains quite low. In addition, reprogramming efficacy has been evidenced to be affected by disease mutations that are present in patient samples. In this study, using RNA-seq platform we have identified and validated the differential gene expression of five transcription factors (TFs) (GBX2, NANOGP8, SP8, PEG3, and ZIC1) that were associated with a remarkable increase in the number of iPSC colonies generated from a patient with Parkinson's disease. We have applied different bioinformatics tools (Gene ontology, protein-protein interaction, and signaling pathways analyses) to investigate the possible roles of these TFs in pluripotency and developmental process. Interestingly, GBX2, NANOGP8, SP8, PEG3, and ZIC1 were found to play a role in maintaining pluripotency, regulating self-renewal stages, and interacting with other factors that are involved in pluripotency regulation including OCT4, SOX2, NANOG, and KLF4. Therefore, the TFs identified in this study could be used as additional transcription factors that enhance reprogramming efficiency to boost iPSC generation technology.
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Affiliation(s)
- Nuha T Swaidan
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation (QF), Doha, Qatar
| | - Salam Salloum-Asfar
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation (QF), Doha, Qatar
| | - Freshteh Palangi
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation (QF), Doha, Qatar
| | - Khaoula Errafii
- Genomics Core Facility, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, Doha, Qatar.,College of Health and Life Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha, Qatar
| | - Nada H Soliman
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Ahmed T Aboughalia
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Abdul Haseeb S Wali
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Sara A Abdulla
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation (QF), Doha, Qatar.
| | - Mohamed M Emara
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar. .,Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, Doha, Qatar.
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24
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Petkov S, Dressel R, Rodriguez-Polo I, Behr R. Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules. Cells 2020; 9:cells9112422. [PMID: 33167468 PMCID: PMC7694496 DOI: 10.3390/cells9112422] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Accepted: 11/03/2020] [Indexed: 12/12/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.
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Affiliation(s)
- Stoyan Petkov
- Platform Degenerative Diseases, German Primate Center, GmbH, Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- German Center for Cardiovascular Research (DZHK), partner site Göttingen, 37077 Göttingen, Germany;
- Correspondence: (S.P.); (R.B.); Tel.: +49-(0)551-3851-322 (S.P.); Tel.:+49-(0)551-3851-132 (R.B.)
| | - Ralf Dressel
- German Center for Cardiovascular Research (DZHK), partner site Göttingen, 37077 Göttingen, Germany;
- Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Ignacio Rodriguez-Polo
- Platform Degenerative Diseases, German Primate Center, GmbH, Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- German Center for Cardiovascular Research (DZHK), partner site Göttingen, 37077 Göttingen, Germany;
| | - Rüdiger Behr
- Platform Degenerative Diseases, German Primate Center, GmbH, Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- German Center for Cardiovascular Research (DZHK), partner site Göttingen, 37077 Göttingen, Germany;
- Correspondence: (S.P.); (R.B.); Tel.: +49-(0)551-3851-322 (S.P.); Tel.:+49-(0)551-3851-132 (R.B.)
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25
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Zhu Z, Zhang Y, Wang X, Wang X, Ye SD. Inhibition of protein kinase D by CID755673 promotes maintenance of the pluripotency of embryonic stem cells. Development 2020; 147:dev185264. [PMID: 32747433 DOI: 10.1242/dev.185264] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Accepted: 07/20/2020] [Indexed: 12/30/2022]
Abstract
The identification of novel mechanisms to maintain embryonic stem cell (ESC) pluripotency is of crucial importance, because the currently used culture conditions are not suitable for ESCs from all species. In this study, we show that the protein kinase D (PKD) inhibitor CID755673 (CID) is able to maintain the undifferentiated state of mouse ESCs in combination with the mitogen-activated protein kinase kinase (MEK) inhibitor. The expression levels of PKD members, including PKD1, PKD2 and PKD3, were low in mouse ESCs but significantly increased under differentiation conditions. Therefore, depletion of three PKD genes was able to phenocopy PKD inhibition. Mechanistically, PKD inhibition activated PI3K/AKT signaling by increasing the level of AKT phosphorylation, and the addition of a PI3K/AKT signaling pathway inhibitor partially reduced the cellular response to PKD inhibition. Importantly, the self-renewal-promoting effect of CID was maintained in human ESCs. Simultaneous knockdown of the three human PKD isoforms enabled short-term self-renewal in human ESCs, whereas PI3K/AKT signaling inhibition eliminated this self-renewal ability downstream of the PKD inhibitor. These findings expand our understanding of the gene regulatory network of ESC pluripotency.
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Affiliation(s)
- Zhenhua Zhu
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, P.R. China
| | - Yan Zhang
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, P.R. China
| | - Xiaoxiao Wang
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, P.R. China
| | - Xiaohu Wang
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, P.R. China
| | - Shou-Dong Ye
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui 230601, P.R. China
- Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, P.R. China
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26
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Yu S, Zhou C, Cao S, He J, Cai B, Wu K, Qin Y, Huang X, Xiao L, Ye J, Xu S, Xie W, Kuang J, Chu S, Guo J, Liu H, Pang W, Guo L, Zeng M, Wang X, Luo R, Li C, Zhao G, Wang B, Wu L, Chen J, Liu J, Pei D. BMP4 resets mouse epiblast stem cells to naive pluripotency through ZBTB7A/B-mediated chromatin remodelling. Nat Cell Biol 2020; 22:651-662. [PMID: 32393886 DOI: 10.1038/s41556-020-0516-x] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2019] [Accepted: 04/03/2020] [Indexed: 01/09/2023]
Abstract
BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.
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Affiliation(s)
- Shengyong Yu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Chunhua Zhou
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Shangtao Cao
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Jiangping He
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Baomei Cai
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Kaixin Wu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China
| | - Yue Qin
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Xingnan Huang
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangzhou Branch of the Supercomputing Center, Chinese Academy of Sciences, Guangzhou, China
| | - Lizhan Xiao
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China
| | - Jing Ye
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Shuyang Xu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Wenxiu Xie
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Junqi Kuang
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Shilong Chu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Jing Guo
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - He Liu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Wei Pang
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Lin Guo
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Mengying Zeng
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Xiaoshan Wang
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China.,Guangzhou Branch of the Supercomputing Center, Chinese Academy of Sciences, Guangzhou, China
| | - Rongping Luo
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Chen Li
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Guoqing Zhao
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of the Chinese Academy of Sciences, Beijing, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China.,Laboratory of Regenerative Biology, Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China
| | - Bo Wang
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Linlin Wu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
| | - Jiekai Chen
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China.,Guangzhou Branch of the Supercomputing Center, Chinese Academy of Sciences, Guangzhou, China.,Laboratory of Regenerative Biology, Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China
| | - Jing Liu
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. .,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. .,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China. .,Guangzhou Branch of the Supercomputing Center, Chinese Academy of Sciences, Guangzhou, China. .,Laboratory of Regenerative Biology, Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China.
| | - Duanqing Pei
- CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. .,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. .,Center for Cell Fate and Lineage, Division of Basic Research and International Corporation, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China. .,Guangzhou Branch of the Supercomputing Center, Chinese Academy of Sciences, Guangzhou, China. .,Laboratory of Regenerative Biology, Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China.
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27
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Xiong C, Wang M, Ling W, Xie D, Chu X, Li Y, Huang Y, Li T, Otieno E, Qiu X, Xiao X. Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro. Cell Reprogram 2020; 22:43-54. [PMID: 32150690 DOI: 10.1089/cell.2019.0080] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.
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Affiliation(s)
- Chunxia Xiong
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Mingyu Wang
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Wenhui Ling
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Dengfeng Xie
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Xinyue Chu
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Yunxin Li
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Yun Huang
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Tong Li
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Edward Otieno
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Xiaoyan Qiu
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
| | - Xiong Xiao
- Department of Veterinary Medicine, College of Animal Science and Technology, Southwest University, Chongqing, China
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Kang X, Li C. Landscape inferred from gene expression data governs pluripotency in embryonic stem cells. Comput Struct Biotechnol J 2020; 18:366-374. [PMID: 32128066 PMCID: PMC7044515 DOI: 10.1016/j.csbj.2020.02.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2019] [Revised: 02/07/2020] [Accepted: 02/09/2020] [Indexed: 12/21/2022] Open
Abstract
Embryonic stem cells (ESCs) can differentiate into diverse cell types and have the ability of self-renewal. Therefore, the study of cell fate decisions on embryonic stem cells has far-reaching significance for regenerative medicine and other biomedical fields. Mathematical models have been used to study emryonic stem cell differentiation. However, the underlying mechanisms of cell differentiation and lineage reprogramming remain to be elucidated. Especially, how to integrate the computational models with quantitative experimental data is still challenging. In this work, we developed a data-constrained modelling approach, and established a model of mouse embryonic stem cells. We used the truncated moment equations (TME) method to quantify the potential landscape of the ESC network. We identified two attractors on the landscape, which represent the embryonic stem cell (ESC) state and differentiated cell (DC) state, respectively, and quantified high dimensional biological paths for differentiation and reprogramming process. Through identifying the optimal combinations of gene targets based on a landscape control strategy, we offered some predictions about the key regulatory factors that govern the differentiation and reprogramming in ESCs.
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Affiliation(s)
- Xin Kang
- School of Mathematical Sciences, Fudan University, Shanghai, China.,Shanghai Center for Mathematical Sciences, Fudan University, Shanghai, China
| | - Chunhe Li
- Shanghai Center for Mathematical Sciences, Fudan University, Shanghai, China.,Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai, China
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Kidder BL. Derivation of LIF-Independent Embryonic Stem Cells Using Inducible OCT4 Expression. Methods Mol Biol 2020; 2117:229-234. [PMID: 31960382 DOI: 10.1007/978-1-0716-0301-7_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Pluripotent mouse embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of preimplantation stage embryos, are capable of self-renewing indefinitely in the presence of the external signal leukemia inhibitory factor (LIF), activation of Wnt signaling through inhibition of GSK3, and inhibition of MAP kinase/ERK kinase signaling. The OCT4 transcription factor is expressed highly in pluripotent cells and is a central transcriptional regulator of the pluripotent state. Here, we describe a protocol to culture ES cells in LIF-independent and serum-free media using an inducible OCT4 (iOCT4) ES cell model system. This protocol is sufficient to sustain ES cell self-renewal in vitro in defined conditions in the absence of external signals. LIF-independent iOCT4 ES cells are fully capable of differentiating following deactivation of the inducible OCT4 transgene.
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Affiliation(s)
- Benjamin L Kidder
- Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA.
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30
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Gordeeva O. TGFβ Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells' Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer. Cells 2019; 8:cells8121500. [PMID: 31771212 PMCID: PMC6953027 DOI: 10.3390/cells8121500] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Revised: 11/19/2019] [Accepted: 11/21/2019] [Indexed: 12/11/2022] Open
Abstract
The transforming growth factor-β (TGFβ) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFβ family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFβ family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFβ family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFβ family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.
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Affiliation(s)
- Olga Gordeeva
- Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov str., 119334 Moscow, Russia
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31
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Gonnot F, Langer D, Bourillot PY, Doerflinger N, Savatier P. Regulation of Cyclin E by transcription factors of the naïve pluripotency network in mouse embryonic stem cells. Cell Cycle 2019; 18:2697-2712. [PMID: 31462142 PMCID: PMC6773236 DOI: 10.1080/15384101.2019.1656475] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Continuous, non-cell cycle-dependent expression of cyclin E is a characteristic feature of mouse embryonic stem cells (mESCs). We studied the 5′ regulatory region of Cyclin E, also known as Ccne1, and identified binding sites for transcription factors of the naïve pluripotency network, including Esrrb, Klf4, and Tfcp2l1 within 1 kilobase upstream of the transcription start site. Luciferase assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChiP–qPCR) study highlighted one binding site for Esrrb that is essential to transcriptional activity of the promoter region, and three binding sites for Klf4 and Tfcp2l1. Knockdown of Esrrb, Klf4, and Tfcp2l1 reduced Cyclin E expression whereas overexpression of Esrrb and Klf4 increased it, indicating a strong correlation between the expression level of these factors and that of cyclin E. We observed that cyclin E overexpression delays differentiation induced by Esrrb depletion, suggesting that cyclin E is an important target of Esrrb for differentiation blockade. We observed that mESCs express a low level of miR-15a and that transfection of a miR-15a mimic decreases Cyclin E mRNA level. These results lead to the conclusion that the high expression level of Cyclin E in mESCs can be attributed to transcriptional activation by Esrrb as well as to the absence of its negative regulator, miR-15a.
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Affiliation(s)
- Fabrice Gonnot
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Diana Langer
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Pierre-Yves Bourillot
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Nathalie Doerflinger
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Pierre Savatier
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
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32
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Protein Kinases and Their Inhibitors in Pluripotent Stem Cell Fate Regulation. Stem Cells Int 2019; 2019:1569740. [PMID: 31428157 PMCID: PMC6681599 DOI: 10.1155/2019/1569740] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 05/31/2019] [Accepted: 06/16/2019] [Indexed: 12/25/2022] Open
Abstract
Protein kinases modulate the reversible postmodifications of substrate proteins to their phosphorylated forms as an essential process in regulating intracellular signaling transduction cascades. Moreover, phosphorylation has recently been shown to tightly control the regulatory network of kinases responsible for the induction and maintenance of pluripotency, defined as the particular ability to differentiate pluripotent stem cells (PSCs) into every cell type in the adult body. In particular, emerging evidence indicates that the balance between the self-renewal and differentiation of PSCs is regulated by the small molecules that modulate kinase signaling pathways. Furthermore, new reprogramming technologies have been developed using kinase modulators, which have provided novel insight of the mechanisms underlying the kinase regulatory networks involved in the generation of induced pluripotent stem cells (iPSCs). In this review, we highlight the recent progress made in defining the roles of protein kinase signaling pathways and their small molecule modulators in regulating the pluripotent states, self-renewal, reprogramming process, and lineage differentiation of PSCs.
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33
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Fang Y, Yuan Y, Zhang LL, Lu JW, Feng JF, Hu SN. Downregulated GBX2 gene suppresses proliferation, invasion and angiogenesis of breast cancer cells through inhibiting the Wnt/β-catenin signaling pathway. Cancer Biomark 2019; 23:405-418. [PMID: 30223390 DOI: 10.3233/cbm-181466] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
OBJECTIVE Gastrulation brain homeobox 2 (GBX2), a gene involved in mid/hindbrain region, has been revealed as one of the oncogene associated with certain cancers, as an example being prostate cancer. However, despite years of worldwide research, the underlying mechanism of GBX2 as well as its significance in breast cancer still remains unclear. Therefore, the present study evaluates the abilities of GBX gene silencing providing for the proliferation, invasion and angiogenesis of breast cancer cells by way of the Wnt/β-catenin signaling pathway. METHODS We employed a microarray analysis to screen out differentially expressed genes relative to breast cancer. Moreover, we retrieved GBX2 expression in breast cancer to find out the relationship between GBX2 expression and prognosis in breast cancer. We performed RT-qPCR to screen out cell lines with high GBX2 expression. Subsequently, both RT-qPCR and western blot analysis were employed so as to measure the combination of the mRNA and protein expressions of GBX2, β-catenin, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. The effect that GBX2 gene silencing and the Wnt/β-catenin signaling pathway had on cell proliferation, invasion, angiogenesis, and tumorigenic ability were evaluated. RESULTS GBX2 gene was also identified having played a role in breast cancer development due to its association with the Wnt/β-catenin signaling pathway. GBX2 gene silencing was found to be an inhibitor for the mRNA and protein expressions regulating β-catenin, VEGF, MMP-2, and MMP-9. Cell proliferation, invasion, angiogenesis, as well as tumorigenic ability in breast cancer were investigated and found to have been suppressed by the GBX2 gene silencing or inactivation of the Wnt/β-catenin signaling pathway. CONCLUSION The study has made an attempt to provide evidence to the idea that GBX2 gene silencing has an inhibition effect on the proliferation, invasion and angiogenesis of the breast cancer cells by inhibiting the activation of the Wnt/β-catenin signaling pathway.
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Whitworth DJ, Limnios IJ, Gauthier ME, Weeratunga P, Ovchinnikov DA, Baillie G, Grimmond SM, Graves JAM, Wolvetang EJ. Platypus Induced Pluripotent Stem Cells: The Unique Pluripotency Signature of a Monotreme. Stem Cells Dev 2019; 28:151-164. [PMID: 30417748 DOI: 10.1089/scd.2018.0179] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted.
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Affiliation(s)
- Deanne J Whitworth
- 1 School of Veterinary Science, University of Queensland, Gatton, Australia.,2 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, Australia
| | - Ioannis J Limnios
- 2 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, Australia.,3 Research School of Biology, Australian National University, Acton, Australia.,4 Clem Jones Centre for Regenerative Medicine, Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland, Australia
| | | | | | - Dmitry A Ovchinnikov
- 2 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, Australia
| | - Gregory Baillie
- 5 Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia
| | - Sean M Grimmond
- 5 Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia
| | | | - Ernst J Wolvetang
- 2 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, Australia
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35
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Abstract
Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irradiated mouse embryonic fibroblasts (iMEFs). This culture system reliably sustains mESC pluripotency and self-renewal capacity, allowing their use in a wide range of experimental settings.
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Affiliation(s)
- Jacob M Paynter
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
| | - Joseph Chen
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
| | - Xiaodong Liu
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
| | - Christian M Nefzger
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia.
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia.
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia.
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
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36
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Abstract
The Reasoning Engine for Interaction Networks (RE:IN) is a tool that was developed initially for the study of pluripotency in mouse embryonic stem cells. A set of critical factors that regulate the pluripotent state had been identified experimentally, but it was not known how these genes interacted to stabilize self-renewal or commit the cell to differentiation. The methodology encapsulated in RE:IN enabled the exploration of a space of possible network interaction models, allowing for uncertainty in whether individual interactions exist between the pluripotency factors. This concept of an "abstract" network was combined with automated reasoning that allows the user to eliminate models that are inconsistent with experimental observations. The tool generalizes beyond the study of stem cell decision-making, allowing for the study of interaction networks more broadly across biology.
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Dunn SJ, Li MA, Carbognin E, Smith A, Martello G. A common molecular logic determines embryonic stem cell self-renewal and reprogramming. EMBO J 2018; 38:embj.2018100003. [PMID: 30482756 PMCID: PMC6316172 DOI: 10.15252/embj.2018100003] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 10/03/2018] [Accepted: 10/04/2018] [Indexed: 11/18/2022] Open
Abstract
During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single‐cell resolution by RT–qPCR. Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.
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Affiliation(s)
- Sara-Jane Dunn
- Microsoft Research, Cambridge, UK.,Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Meng Amy Li
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Elena Carbognin
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Austin Smith
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK .,Department of Biochemistry, University of Cambridge, Cambridge, UK
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Zhang Y, Wang D, Xu J, Wang Y, Ma F, Li Z, Liu N. Stat3 activation is critical for pluripotency maintenance. J Cell Physiol 2018; 234:1044-1051. [DOI: 10.1002/jcp.27241] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 07/25/2018] [Indexed: 12/28/2022]
Affiliation(s)
- Yan Zhang
- Department of Cell Biology School of Medicine, Nankai University Tianjin China
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
| | - Dan Wang
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
- Department of Genetics and Cell Biology College of Life Sciences, Nankai University Tianjin China
| | - Jia Xu
- Department of Cell Biology School of Medicine, Nankai University Tianjin China
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
| | - Yuebing Wang
- Department of Cell Biology School of Medicine, Nankai University Tianjin China
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
| | - Fengxia Ma
- State Key Lab of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences Tianjin China
| | - Zongjin Li
- Department of Cell Biology School of Medicine, Nankai University Tianjin China
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
| | - Na Liu
- Department of Cell Biology School of Medicine, Nankai University Tianjin China
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University Tianjin China
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Pickering J, Rich CA, Stainton H, Aceituno C, Chinnaiya K, Saiz-Lopez P, Ros MA, Towers M. An intrinsic cell cycle timer terminates limb bud outgrowth. eLife 2018; 7:37429. [PMID: 30175958 PMCID: PMC6143340 DOI: 10.7554/elife.37429] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Accepted: 09/02/2018] [Indexed: 12/24/2022] Open
Abstract
The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.
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Affiliation(s)
- Joseph Pickering
- Department of Biomedical Science, The Bateson Centre, University of Sheffield, Sheffield, United Kingdom
| | - Constance A Rich
- Department of Biomedical Science, The Bateson Centre, University of Sheffield, Sheffield, United Kingdom
| | - Holly Stainton
- Department of Biomedical Science, The Bateson Centre, University of Sheffield, Sheffield, United Kingdom
| | - Cristina Aceituno
- Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-Universidad de Cantabria), Santander, Spain
| | - Kavitha Chinnaiya
- Department of Biomedical Science, The Bateson Centre, University of Sheffield, Sheffield, United Kingdom
| | - Patricia Saiz-Lopez
- Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-Universidad de Cantabria), Santander, Spain
| | - Marian A Ros
- Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-Universidad de Cantabria), Santander, Spain.,Departamento de Anatomía y Biología Celular, Facultad de Medicina, Universidad de Cantabria, Santander, Spain
| | - Matthew Towers
- Department of Biomedical Science, The Bateson Centre, University of Sheffield, Sheffield, United Kingdom
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Yamane M, Ohtsuka S, Matsuura K, Nakamura A, Niwa H. Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells. Development 2018; 145:dev.162404. [PMID: 29739838 DOI: 10.1242/dev.162404] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2017] [Accepted: 04/30/2018] [Indexed: 01/02/2023]
Abstract
Krüppel-like factors (Klfs) have a pivotal role in maintaining self-renewal of mouse embryonic stem cells (mESCs). The functions of three Klf family members (Klf2, Klf4 and Klf5) have been identified, and are suggested to largely overlap. For further dissection of their functions, we applied an inducible knockout system for these Klf family members and assessed the effects of combinatorial loss of function. As a result, we confirmed that any one of Klf2, Klf4 and Klf5 was sufficient to support self-renewal, whereas the removal of all three compromised it. The activity of any single transcription factor, except for a Klf family member, was not sufficient to restore self-renewal of triple-knockout mESCs. However, some particular combinations of transcription factors were capable of the restoration. The triple-knockout mESCs were successfully captured at primed state. These data indicate that the pivotal function of a Klf family member is transduced into the activation of multiple transcription factors in a naïve-state-specific manner.
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Affiliation(s)
- Mariko Yamane
- Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.,Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
| | - Satoshi Ohtsuka
- Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.,Department of Life Science, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada kahoku, Ishikawa 920-0293, Japan
| | - Kumi Matsuura
- Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.,Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
| | - Akira Nakamura
- Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
| | - Hitoshi Niwa
- Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan .,Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.,JST, CREST, Sanbancho, Chiyoda-ku, Tokyo 1020075, Japan
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41
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Papatsenko D, Waghray A, Lemischka IR. Feedback control of pluripotency in embryonic stem cells: Signaling, transcription and epigenetics. Stem Cell Res 2018; 29:180-188. [DOI: 10.1016/j.scr.2018.02.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Revised: 02/06/2018] [Accepted: 02/16/2018] [Indexed: 12/19/2022] Open
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42
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Abstract
Tissue-specific transcription factors primarily act to define the phenotype of the cell. The power of a single transcription factor to alter cell fate is often minimal, as seen in gain-of-function analyses, but when multiple transcription factors cooperate synergistically it potentiates their ability to induce changes in cell fate. By contrast, transcription factor function is often dispensable in the maintenance of cell phenotype, as is evident in loss-of-function assays. Why does this phenomenon, commonly known as redundancy, occur? Here, I discuss the role that transcription factor networks play in collaboratively regulating stem cell fate and differentiation by providing multiple explanations for their functional redundancy.
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Affiliation(s)
- Hitoshi Niwa
- Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
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43
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Ying QL, Smith A. The Art of Capturing Pluripotency: Creating the Right Culture. Stem Cell Reports 2018; 8:1457-1464. [PMID: 28591647 PMCID: PMC5470336 DOI: 10.1016/j.stemcr.2017.05.020] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2017] [Revised: 05/17/2017] [Accepted: 05/17/2017] [Indexed: 12/14/2022] Open
Abstract
Embryonic stem cells (ESCs) are a unique tool for genetic perturbation of mammalian cellular and organismal processes additionally in humans offer unprecedented opportunities for disease modeling and cell therapy. Furthermore, ESCs are a powerful system for exploring the fundamental biology of pluripotency. Indeed understanding the control of self-renewal and differentiation is key to realizing the potential of ESCs. Building on previous observations, we found that mouse ESCs can be derived and maintained with high efficiency through insulation from differentiation cues combined with consolidation of an innate cell proliferation program. This finding of a pluripotent ground state has led to conceptual and practical advances, including the establishment of germline-competent ESCs from recalcitrant mouse strains and for the first time from the rat. Here, we summarize historical and recent progress in defining the signaling environment that supports self-renewal. We compare the contrasting requirements of two types of pluripotent stem cell, naive ESCs and primed post-implantation epiblast stem cells (EpiSCs), and consider the outstanding challenge of generating naive pluripotent stem cells from different mammals.
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Affiliation(s)
- Qi-Long Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
| | - Austin Smith
- Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
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44
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Yachie-Kinoshita A, Onishi K, Ostblom J, Langley MA, Posfai E, Rossant J, Zandstra PW. Modeling signaling-dependent pluripotency with Boolean logic to predict cell fate transitions. Mol Syst Biol 2018; 14:e7952. [PMID: 29378814 PMCID: PMC5787708 DOI: 10.15252/msb.20177952] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The mechanisms that maintain pluripotency, or that cause its destabilization to initiate development, are complex and incompletely understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to input signals. Our strategy used random asynchronous Boolean simulations (R-ABS) to simulate single-cell fate transitions and strongly connected components (SCCs) strategy to represent population heterogeneity. This framework was applied to a reverse-engineered and curated core GRN for mouse embryonic stem cells (mESCs) and used to simulate cellular responses to combinations of five signaling pathways. Our simulations predicted experimentally verified cell population compositions and input signal combinations controlling specific cell fate transitions. Extending the model to PSC differentiation, we predicted a combination of signaling activators and inhibitors that efficiently and robustly generated a Cdx2+Oct4- cells from naïve mESCs. Overall, this platform provides new strategies to simulate cell fate transitions and the heterogeneity that typically occurs during development and differentiation.
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Affiliation(s)
- Ayako Yachie-Kinoshita
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.,The Donnelly Centre, University of Toronto, Toronto, ON, Canada.,The Systems Biology Institute, Minato, Tokyo, Japan
| | - Kento Onishi
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.,The Donnelly Centre, University of Toronto, Toronto, ON, Canada
| | - Joel Ostblom
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.,The Donnelly Centre, University of Toronto, Toronto, ON, Canada
| | - Matthew A Langley
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.,The Donnelly Centre, University of Toronto, Toronto, ON, Canada
| | - Eszter Posfai
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, ON, Canada
| | - Janet Rossant
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, ON, Canada
| | - Peter W Zandstra
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada .,The Donnelly Centre, University of Toronto, Toronto, ON, Canada.,Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada.,Medicine by Design, A Canada First Research Excellence Program at the University of Toronto, Toronto, ON, Canada
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45
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Kinoshita M, Smith A. Pluripotency Deconstructed. Dev Growth Differ 2018; 60:44-52. [PMID: 29359419 DOI: 10.1111/dgd.12419] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2017] [Accepted: 12/02/2017] [Indexed: 12/14/2022]
Abstract
Pluripotency denotes the flexible capacity of single cells to give rise to all somatic lineages and typically also the germline. Mouse ES cells and post-implantation epiblast-derived stem cells (EpiSC) are widely used pluripotent cell culture systems. These two in vitro stem cell types have divergent characteristics. They are considered as representative of distinct developmental stages, distinguished by using the terms "naïve" and "primed". A binary description is an over-simplification, however. Here, we discuss an intermediate stage of pluripotency that we term "formative". Formative pluripotency features a gene regulatory network switch from the naïve state and comprises capacitation of enhancers, signaling pathways and epigenetic machinery in order to install competence for lineage specification.
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Affiliation(s)
- Masaki Kinoshita
- Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge, UK
| | - Austin Smith
- Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge, UK.,Department of Biochemistry, University of Cambridge, Cambridge, UK
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46
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Huang D, Wang L, Duan J, Huang C, Tian XC, Zhang M, Tang Y. LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming. Biol Open 2018; 7:bio.029264. [PMID: 29212799 PMCID: PMC5829498 DOI: 10.1242/bio.029264] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. Summary:Esrrb is a downstream target and effector of LIF during reprogramming. Forced Esrrb expression accelerates pluripotency establishment in the absence of LIF signaling. The activation of Esrrb is LIF dependent in the reprogramming process.
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Affiliation(s)
- Delun Huang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi 530004, China.,Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Ling Wang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Jingyue Duan
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Chang Huang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Xiuchun Cindy Tian
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Ming Zhang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi 530004, China
| | - Young Tang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
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47
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Wei J, Fan Z, Yang Z, Zhou Y, Da F, Zhou L, Tao W, Wang D. Leukemia Inhibitory Factor Is Essential for the Self-Renewal of Embryonic Stem Cells from Nile Tilapia (Oreochromis niloticus) Through Stat3 Signaling. Stem Cells Dev 2018; 27:123-132. [DOI: 10.1089/scd.2017.0207] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Affiliation(s)
- Jing Wei
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Zhenhua Fan
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Zhuo Yang
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Yujie Zhou
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Fan Da
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Linyan Zhou
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Wenjing Tao
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Deshou Wang
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
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48
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He R, Xhabija B, Al-Qanber B, Kidder BL. OCT4 supports extended LIF-independent self-renewal and maintenance of transcriptional and epigenetic networks in embryonic stem cells. Sci Rep 2017; 7:16360. [PMID: 29180818 PMCID: PMC5703885 DOI: 10.1038/s41598-017-16611-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Accepted: 11/15/2017] [Indexed: 12/29/2022] Open
Abstract
Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.
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Affiliation(s)
- Runsheng He
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA.,Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA
| | - Besa Xhabija
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA.,Department of Chemistry and Biochemistry, University of Michigan-Flint, Flint, MI, USA
| | - Batool Al-Qanber
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA.,Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA
| | - Benjamin L Kidder
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA. .,Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA.
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49
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Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. Nat Commun 2017; 8:1096. [PMID: 29061959 PMCID: PMC5653659 DOI: 10.1038/s41467-017-01076-4] [Citation(s) in RCA: 122] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Accepted: 08/15/2017] [Indexed: 01/01/2023] Open
Abstract
Gene expression heterogeneity in the pluripotent state of mouse
embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast,
exit from pluripotency and lineage commitment have not been studied systematically
at the single-cell level. Here we measure the gene expression dynamics of retinoic
acid driven mESC differentiation from pluripotency to lineage commitment, using an
unbiased single-cell transcriptomics approach. We find that the exit from
pluripotency marks the start of a lineage transition as well as a transient phase of
increased susceptibility to lineage specifying signals. Our study reveals several
transcriptional signatures of this phase, including a sharp increase of gene
expression variability and sequential expression of two classes of transcriptional
regulators. In summary, we provide a comprehensive analysis of the exit from
pluripotency and lineage commitment at the single cell level, a potential stepping
stone to improved lineage manipulation through timing of differentiation
cues. Commitment to different fates by differentiating pluripotent cells
depends upon integration of external and internal signals. Here the authors analyse
the entry of mouse embryonic stem cells into retinoic acid-mediated differentiation
using single cell transcriptomics with high temporal resolution.
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50
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Tang L, Wang M, Liu D, Gong M, Ying QL, Ye S. Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal. PLoS One 2017; 12:e0185714. [PMID: 28961274 PMCID: PMC5621696 DOI: 10.1371/journal.pone.0185714] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Accepted: 09/18/2017] [Indexed: 11/22/2022] Open
Abstract
Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of Sp5 induced Nanog expression, while knockdown or knockout of Sp5 decreased the Nanog level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of Nanog eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.
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Affiliation(s)
- Ling Tang
- Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China
| | - Manman Wang
- Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China
| | - Dahai Liu
- Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China
| | - Mengting Gong
- Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China
| | - Qi-Long Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America
| | - Shoudong Ye
- Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China
- * E-mail:
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