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Zonooz ER, Ghezelayagh Z, Moradmand A, Aghayan HR, Shekari F, Tahamtani Y. Potential role of Sigma-1 receptor inhibition and ER stress-related pathways in upregulating definitive endoderm markers in human embryonic stem cells. Exp Cell Res 2025; 448:114557. [PMID: 40221006 DOI: 10.1016/j.yexcr.2025.114557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 03/03/2025] [Accepted: 04/10/2025] [Indexed: 04/14/2025]
Abstract
Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) participate in stem cell proliferation, differentiation, and apoptosis. Sigma-1 receptor (S1R) is a unique ER chaperon protein that regulates ER stress and UPR. Here, we examine the effects of S1R inhibition on pluripotency and differentiation of human embryonic stem cells (hESCs). hESCs were treated with different doses of an S1R inhibitor (BD 1047), and we investigated the expressions of different pluripotency and lineage-specific genes. The BD-treated hESCs showed increased SRY-box transcription factor 17 (SOX17) expression [definitive endoderm-specific protein], and reductions in NANOG expression and in the number of alkaline phosphatase (ALP)-positive colonies. In silico gene expression analysis of three datasets that contained the hESCs-derived DE samples (GSE98324, GSE63592, GSE52658) was performed to investigate the ER stress-related gene expression patterns during DE differentiation. In silico analysis revealed that UPR-related genes upregulated during DE differentiation and CCL2 was the only gene present in all three DE datasets. qRT-PCR and immunostaining showed that CCL2, eIF2A, ATF4, XBP1, GRP78, DDIT3, DNAJB9, and PDIA5 which are UPR related marker genes were all upregulated in both the BD-treated hESCs and female and male hESC-derived DE cells. The results of this study suggest possible roles for S1R, ER stress-related genes, and the CCL2 pathway during differentiation of hESCs into DE. These potential new targets may improve the efficiency of protocols used to differentiate endodermal lineages.
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Affiliation(s)
- Elmira Rezaei Zonooz
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Zahra Ghezelayagh
- Department of Basic and Population-based Studies in NCD, Reproductive Epidemiology Research Center, Royan Institute, ACECR, Tehran, Iran
| | - Azadeh Moradmand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Advanced Therapy Medicinal Product Technology Development Center (ATMP-TDC), Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hamid Reza Aghayan
- Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Faezeh Shekari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - Yaser Tahamtani
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Basic and Population-based Studies in NCD, Reproductive Epidemiology Research Center, Royan Institute, ACECR, Tehran, Iran.
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2
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Huang W, Hill JC, Patel S, Richards TD, Sultan I, Kaczorowski DJ, Phillippi JA. Deficiency of fibroblast growth factor 2 promotes contractile phenotype of pericytes in ascending thoracic aortic aneurysm. Am J Physiol Heart Circ Physiol 2025; 328:H1130-H1143. [PMID: 40214073 DOI: 10.1152/ajpheart.00834.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 12/31/2024] [Accepted: 03/20/2025] [Indexed: 05/01/2025]
Abstract
Pericytes exhibit progenitor cell-like qualities and associate with the vasa vasorum-vital microvessels nourishing larger arteries and veins. How pericytes change in human ascending thoracic aortic aneurysm (ATAA) remains unknown. Here, we used the public single-nuclei sequencing data to reveal a contractile phenotype transition of pericytes in human ATAA specimens. In addition, we found that a protective factor, fibroblast growth factor 2 (FGF2), is decreased in the aortic adventitia of both male and female patients with ATAA and impacts pericytes. We demonstrated that FGF2 maintained pericytes in a less contractile and high angiogenic phenotype via MAPK and PI3K-AKT signaling pathways. These findings suggested the latent engagement of pericytes in ATAA, providing insights that could guide the development of new therapies against aortic disease.NEW & NOTEWORTHY Here, we revealed that pericytes transition into a contractile phenotype in human ATAA. We demonstrated that FGF2 maintained pericytes in a less contractile and high angiogenic stage via MAPK and PI3K-AKT signaling pathway, whereas we found FGF2 is decreased in the aortic adventitia of patients with ATAA. Our findings suggest how growth factor deficiency in the microenvironment affects pericytes during ATAA, offering leads for potential new therapies for aortic diseases.
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Affiliation(s)
- Weijian Huang
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Department of Cardiac Surgery, Xiangya Hospital, Central South University, Changsha, People's Republic of China
| | - Jennifer C Hill
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
| | - Sakshi Patel
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
| | - Tara D Richards
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
| | - Ibrahim Sultan
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Heart and Vascular Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States
| | - David J Kaczorowski
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Heart and Vascular Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States
| | - Julie A Phillippi
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
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3
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Zhu X, Huang L, Li G, Deng B, Wang X, Yang H, Zhang Y, Wen Q, Wang C, Zhang J, Zhao Y, Li K, Liu Y. Genome-Wide Silencer Screening Reveals Key Silencer Modulating Reprogramming Efficiency in Mouse Induced Pluripotent Stem Cells. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2408839. [PMID: 40112175 PMCID: PMC12079485 DOI: 10.1002/advs.202408839] [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: 07/30/2024] [Revised: 02/07/2025] [Indexed: 03/22/2025]
Abstract
The majority of the mouse genome is composed of non-coding regions, which harbor numerous regulatory sequences essential for gene regulation. While extensive research focuses on enhancers that activate gene expression, the role of silencers that repress gene expression remains less explored. In this study, the first genome-wide identification of silencers in the mouse genome is conducted. In mouse embryonic fibroblasts (MEFs) and embryonic stem cells (mESCs), 89 596 and 115 165 silencers are identified, respectively. These silencers are ubiquitously distributed across the genome and are predominantly associated with low-expression genes. Additionally, these silencers are mainly cell-specific and function by binding to repressive transcription factors (TFs). Further, these silencers are notably enriched with the histone modification H3K9me3. It is observed that the transformation between dual-function silencers and enhancers is correlated with intracellular transcription factor concentrations, accompanied by changes in epigenetic modifications. In terms of biological effects, we have identified silencers that can enhance the induction efficiency of MEFs and influence the pluripotency of mESCs. Collectively, this work offers the first comprehensive silencer landscape in the mouse genome and provides strong evidence for the role of silencers in the induction of induced pluripotent stem cells (iPSCs).
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Affiliation(s)
- Xiusheng Zhu
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Lei Huang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Guoli Li
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Biao Deng
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
- State Key Laboratory of Genetic Resources and EvolutionYunnan Laboratory of Molecular Biology of Domestic AnimalsKunming Institute of ZoologyChinese Academy of SciencesKunming650201China
- Kunming College of Life ScienceUniversity of Chinese Academy of SciencesKunming650204China
| | - Xiaoxiao Wang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Hu Yang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Yuanyuan Zhang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Qiuhan Wen
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Chao Wang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Jingshu Zhang
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Yunxiang Zhao
- Guangxi Key Laboratory of Animal BreedingDisease Control and PreventionCollege of Animal Science and TechnologyGuangxi UniversityNanning530004China
| | - Kui Li
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
| | - Yuwen Liu
- Shenzhen BranchGuangdong Laboratory of Lingnan Modern AgricultureKey Laboratory of Livestock and Poultry Multi‐omics of MARAAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen518124China
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Torii S, Nagaharu K, Nakanishi N, Usui H, Hori Y, Hirose K, Toyosawa S, Morii E, Narushima M, Kubota Y, Nakagawa O, Imanaka-Yoshida K, Maruyama K. Embryological cellular origins and hypoxia-mediated mechanisms in PIK3CA-driven refractory vascular malformations. EMBO Mol Med 2025:10.1038/s44321-025-00235-1. [PMID: 40234712 DOI: 10.1038/s44321-025-00235-1] [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: 02/13/2025] [Revised: 03/23/2025] [Accepted: 03/27/2025] [Indexed: 04/17/2025] Open
Abstract
Congenital vascular malformations, affecting 0.5% of the population, often occur in the head and neck, complicating treatment due to the critical functions in these regions. Our previous research identified distinct developmental origins for blood and lymphatic vessels in these areas, tracing them to the cardiopharyngeal mesoderm (CPM), which contributes to the development of the head, neck, and cardiovascular system in both mouse and human embryos. In this study, we investigated the pathogenesis of these malformations by expressing Pik3caH1047R in the CPM. Mice expressing Pik3caH1047R in the CPM developed vascular abnormalities restricted to the head and neck. Single-cell RNA sequencing revealed that Pik3caH1047R upregulates Vegf-a expression in endothelial cells through HIF-mediated hypoxia signaling. Human samples supported these findings, showing elevated HIF-1α and VEGF-A in malformed vessels. Notably, inhibition of HIF-1α and VEGF-A in the mouse model significantly reduced abnormal vasculature. These results highlight the role of embryonic origins and hypoxia-driven mechanisms in vascular malformations, providing a foundation for the development of therapies targeting these difficult-to-treat conditions.
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Affiliation(s)
- Sota Torii
- Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan
| | - Keiki Nagaharu
- Department of Hematology and Oncology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, 514-8507, Japan
| | - Nanako Nakanishi
- Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan
| | - Hidehito Usui
- Department of Surgery, Kanagawa Children's Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Kanagawa, Japan
| | - Yumiko Hori
- Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
- Department of Central Laboratory and Surgical Pathology, NHO Osaka National Hospital, 2-1-14 Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan
| | - Katsutoshi Hirose
- Department of Oral and Maxillofacial Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Satoru Toyosawa
- Department of Oral and Maxillofacial Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Eiichi Morii
- Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Mitsunaga Narushima
- Department of Plastic and Reconstructive Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan
| | - Yoshiaki Kubota
- Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Osamu Nakagawa
- Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-shimmachi, Suita, Osaka, 564-8565, Japan
| | - Kyoko Imanaka-Yoshida
- Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan
| | - Kazuaki Maruyama
- Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
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5
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Okubo C, Nakamura M, Sato M, Shichino Y, Mito M, Takashima Y, Iwasaki S, Takahashi K. EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency. SCIENCE ADVANCES 2025; 11:eadq5484. [PMID: 40203091 PMCID: PMC11980838 DOI: 10.1126/sciadv.adq5484] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/19/2024] [Accepted: 03/04/2025] [Indexed: 04/11/2025]
Abstract
Although pluripotent stem cell (PSC) properties, such as differentiation and infinite proliferation, have been well documented within the frameworks of transcription factor networks, epigenomes, and signal transduction, they remain unclear and fragmented. Directing attention toward translational regulation as a bridge between these events can yield additional insights into previously unexplained mechanisms. Our functional CRISPR interference screen-based approach revealed that EIF3D, a translation initiation factor, is crucial for maintaining primed pluripotency. Loss of EIF3D disrupted the balance of pluripotency-associated signaling pathways, thereby compromising primed pluripotency. Moreover, EIF3D ensured robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, EIF3D-mediated translation contributes to tuning the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. This study provides further insights into the translation network in maintaining pluripotency.
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Affiliation(s)
- Chikako Okubo
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| | - Michiko Nakamura
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| | - Masae Sato
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| | - Yuichi Shichino
- RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Saitama, 351-0198, Japan
| | - Mari Mito
- RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Saitama, 351-0198, Japan
| | - Yasuhiro Takashima
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| | - Shintaro Iwasaki
- RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Saitama, 351-0198, Japan
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 277-8561, Japan
| | - Kazutoshi Takahashi
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
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Ziojła NM, Socha M, Guerra MC, Kizewska D, Blaszczyk K, Urbaniak E, Henry S, Grabowska M, Niakan KK, Warmflash A, Borowiak M. ETVs dictate hPSC differentiation by tuning biophysical properties. Nat Commun 2025; 16:1999. [PMID: 40011454 PMCID: PMC11865489 DOI: 10.1038/s41467-025-56591-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Accepted: 01/20/2025] [Indexed: 02/28/2025] Open
Abstract
Stem cells maintain a dynamic dialog with their niche, integrating biochemical and biophysical cues to modulate cellular behavior. Yet, the transcriptional networks that regulate cellular biophysical properties remain poorly defined. Here, we leverage human pluripotent stem cells (hPSCs) and two morphogenesis models - gastruloids and pancreatic differentiation - to establish ETV transcription factors as critical regulators of biophysical parameters and lineage commitment. Genetic ablation of ETV1 or ETV1/ETV4/ETV5 in hPSCs enhances cell-cell and cell-ECM adhesion, leading to aberrant multilineage differentiation including disrupted germ-layer organization, ectoderm loss, and extraembryonic cell overgrowth in gastruloids. Furthermore, ETV1 loss abolishes pancreatic progenitor formation. Single-cell RNA sequencing and follow-up assays reveal dysregulated mechanotransduction via the PI3K/AKT signaling. Our findings highlight the importance of transcriptional control over cell biophysical properties and suggest that manipulating these properties may improve in vitro cell and tissue engineering strategies.
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Affiliation(s)
- Natalia M Ziojła
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Magdalena Socha
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | | | - Dorota Kizewska
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Katarzyna Blaszczyk
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Edyta Urbaniak
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Sara Henry
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Malgorzata Grabowska
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Kathy K Niakan
- The Loke Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | - Aryeh Warmflash
- Department of Biosciences, Rice University, Houston, TX, USA
| | - Malgorzata Borowiak
- Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland.
- McNair Medical Institute, Baylor College of Medicine, Houston, TX, USA.
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Chen W, Chen X, Chen C, She S, Li X, Shan L, Zhang X, Dan S, Wang Y, Zhou YW, Cao Q, Wang W, Hu J, Wei Y, Xue Y, Zhang Y, Zhang S, Wang YJ, Kang B. OCT4 translationally promotes AKT signaling as an RNA-binding protein in stressed pluripotent stem cells. Stem Cell Res Ther 2025; 16:84. [PMID: 39988663 PMCID: PMC11849194 DOI: 10.1186/s13287-025-04229-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Accepted: 02/11/2025] [Indexed: 02/25/2025] Open
Abstract
BACKGROUND Despite numerous studies addressing the molecular mechanisms by which pluripotent stem cells (PSCs) maintain self-renewal and pluripotency under normal culture conditions, the fundamental question of how PSCs manage to survive stressful conditions remains largely unresolved. Post-transcriptional/translational regulation emerges to be vital for PSCs, but how PSCs coordinate and balance their survival and differentiation at translational level under extrinsic and intrinsic stress conditions is unclear. METHODS The high-throughput sequencing of cross-linking immunoprecipitation cDNA library (HITS-CLIP) was employed to decipher the genome-wide OCT4-RNA interactome in human PSCs, a combined RNC-seq/RNA-seq analysis to assess the role of OCT4 in translational regulation of hypoxic PSCs, and an OCT4-protein interactome to search for OCT4 binding partners that regulate cap-independent translation initiation. By taking the Heterozygous Knocking In N-terminal Tags (HKINT) approach that specifically disrupts the 5'-UTR secondary structure and tagging its protein product of the mRNA from one allele while leaving that from the other allele intact, we examined the effect of disrupting the OCT4/5'-UTR interaction on translation of AKT1 mRNA. RESULTS We revealed OCT4 as a bona fide RNA-binding protein (RBP) in human PSCs that bound to the 5'-UTR, 3'-UTR and CDS regions of mRNAs. Multiple known proteins participating in IRES-mediated translation initiation were detected in the OCT4-protein interactome, and a combined RNC-seq/RNA-seq analysis further confirmed a crucial role of OCT4 in translational regulation of PSCs in response to hypoxic stress. Remarkably, OCT4 bound to the GC-rich elements in the 5'-UTR of AKT1 and multiple PI3K/AKT-pathway-gene mRNAs, and promoted their translation initiation via IRES-mediated pathways under stress conditions. Specifically disrupting the AKT1 mRNA 5'-UTR structure and the OCT4/5'-UTR interaction by the HKINT approach significantly reduced the translation level of AKT1 that led to a higher susceptibility of PSCs to oxidative stress-induced apoptotic death and prioritized differentiation toward ectoderm and endoderm. CONCLUSIONS Our results reveal OCT4 as an anti-stress RBP for translational regulation that critically coordinates the survival and differentiation of PSCs in response to various stressors.
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Affiliation(s)
- Wenjie Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
- Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, Zhejiang, China
| | - Xinyu Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
| | - Cheng Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
- Shaoxing People's Hospital, Shaoxing Hospital, Zhejiang University School of Medicine, Shaoxing, 312000, Zhejiang, China
| | - Shiqi She
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
- Zhejiang Museum of Natural History, Hangzhou, 310014, Zhejiang, China
| | - Xia Li
- Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, Zhejiang, China
| | - Lina Shan
- Department of Colorectal SurgerySir Run Run Shaw Hospital,, School of Medicine, Zhejiang University, Hangzhou, 310016, Zhejiang, China
| | - Xiaobing Zhang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
| | - Songsong Dan
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
| | - Yisha Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
| | - Yan-Wen Zhou
- Department of Infectious Diseases, the Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan, China
| | - Qingyi Cao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China
| | - Wenxin Wang
- School of Medicine, Zhejiang University, Hangzhou, 310058, Zhejiang, China
| | - Jianwen Hu
- Shanghai Bioprofile Technology Co., Ltd., Shanghai, 200241, China
| | - Yaxun Wei
- Center for Genome Analysis, ABLife Inc., Wuhan, 430075, Hubei, China
| | - Yaqiang Xue
- Center for Genome Analysis, ABLife Inc., Wuhan, 430075, Hubei, China
| | - Yi Zhang
- Center for Genome Analysis, ABLife Inc., Wuhan, 430075, Hubei, China
| | - Songying Zhang
- Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, Zhejiang, China.
| | - Ying-Jie Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China.
- Cancer Center, Zhejiang University, Hangzhou, 310058, Zhejiang, China.
| | - Bo Kang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang, China.
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8
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Iluta S, Nistor M, Buruiana S, Dima D. Wnt Signaling Pathway in Tumor Biology. Genes (Basel) 2024; 15:1597. [PMID: 39766864 PMCID: PMC11675244 DOI: 10.3390/genes15121597] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2024] [Revised: 12/02/2024] [Accepted: 12/10/2024] [Indexed: 01/11/2025] Open
Abstract
Relapse and metastasis are the major challenges that stand in the way of cancer healing and survival, mainly attributed to cancer stem cells (CSCs). Their capabilities of self-renewal and tumorigenic potential leads to treatment resistance development. CSCs function through signaling pathways such as the Wnt/β-catenin cascade. While commonly involved in embryogenesis and adult tissues homeostasis, the dysregulation of the Wnt pathway has direct correlations with tumorigenesis, metastasis, and drug resistance. The development of therapies that target CSCs and bulk tumors is both crucial and urgent. However, the extensive crosstalk present between Wnt and other signaling networks (Hedgehog and Notch) complicates the development of efficient long-term therapies with minimal side-effects on normal tissues. Despite the obstacles, the emergence of Wnt inhibitors and subsequent modulation of the signaling pathways would provide dynamic therapeutic approaches to impairing CSCs and reversing resistance mechanisms.
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Affiliation(s)
- Sabina Iluta
- Department of Hematology, Iuliu Hatieganu University of Medicine and Pharmacy, 400347 Cluj Napoca, Romania;
| | - Madalina Nistor
- Medfuture Research Center for Advanced Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, 400347 Cluj Napoca, Romania
| | - Sanda Buruiana
- Department of Hematology, Nicolae Testemitanu University of Medicine and Pharmacy, 2004 Chisinau, Moldova;
| | - Delia Dima
- Department of Hematology, Ion Chiricuta Oncology Institute, 400015 Cluj Napoca, Romania
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9
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Chung PH, Lin FH, Liu IH. Enhancing intrinsic TGF-β signaling via heparan sulfate glycosaminoglycan regulation to promote mesenchymal stem cell capabilities and chondrogenesis for cartilage repair. Int J Biol Macromol 2024; 282:137242. [PMID: 39505166 DOI: 10.1016/j.ijbiomac.2024.137242] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Revised: 10/21/2024] [Accepted: 11/02/2024] [Indexed: 11/08/2024]
Abstract
Osteoarthritis burdens patients due to the limited regenerative capacity of chondrocytes. Traditional cartilage repair often falls short, necessitating innovative approaches. Mesenchymal stem cells (MSCs) show promise for regeneration. Heparan sulfate glycosaminoglycans (HS-GAGs) regulate cellular functions, making them a target for cartilage repair. This study highlights how Heparinase III (HepIII) cleaves intact HS-GAGs in bone marrow-derived MSCs (BM-MSCs), enhancing their capabilities and specifically promoting chondrogenesis. HepIII-treated BM-MSCs cultured in a hanging drop device for three days, significantly increased cell number and aggregation into a cell sphere with early chondrogenesis. HepIII promoted BM-MSCs toward chondrogenesis, increasing type II collagen, intact HS-GAGs, and sulfated GAG content, while upregulating chondrogenic and heparan sulfate proteoglycan genes. Treatment with the TGF-β inhibitor (SB-431542) in HepIII-treated BM-MSCs demonstrated enhanced intrinsic transforming growth factor-β (TGF-β) signaling and fibronectin expression. This approach also boosted BM-MSC self-renewal, immunosuppressive potential, and modified acetylated histone signatures, offering a cost-effective strategy for cartilage repair by addressing inflammation, metabolic changes, and the high costs of traditional TGF-β methods. From the results, HepIII-treated BM-MSCs show potential for use in combination with other biopolymers as injectable gels to improve cartilage repair in osteoarthritis patients in the near future.
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Affiliation(s)
- Pei-Hsuan Chung
- Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan.
| | - Feng-Huei Lin
- Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 106, Taiwan; Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli county 350, Taiwan.
| | - I-Hsuan Liu
- Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan; Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei 106, Taiwan.
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10
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Jiang N, Hu Z, Wang Q, Hao J, Yang R, Jiang J, Wang H. Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation. J Cell Physiol 2024; 239:e31423. [PMID: 39188080 DOI: 10.1002/jcp.31423] [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: 03/29/2024] [Revised: 08/01/2024] [Accepted: 08/13/2024] [Indexed: 08/28/2024]
Abstract
Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.
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Affiliation(s)
- Nizhou Jiang
- Department of Spine Surgery, Central Hospital of Dalian University of Technology, Dalian, China
- Department of Spine Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, China
| | - Zhenxin Hu
- Department of Spine Surgery, Peking University Fourth School of Clinical Medicine, Beijing Jishuitan Hospital, Beijing, China
| | - Quanxiang Wang
- Department of Otorhinolaryngology-Head and Neck Surgery, Hongqi Hospital Affiliated to Mudanjiang Medical University, Mudanjiang, China
| | - Jiayu Hao
- Department of Spine Surgery, Central Hospital of Dalian University of Technology, Dalian, China
| | - Rui Yang
- Department of Spine Surgery, Central Hospital of Dalian University of Technology, Dalian, China
| | - Jian Jiang
- Department of Spine Surgery, Central Hospital of Dalian University of Technology, Dalian, China
| | - Hong Wang
- Department of Spine Surgery, Central Hospital of Dalian University of Technology, Dalian, China
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11
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Massafret O, Barragán M, Álvarez-González L, Aran B, Martín-Mur B, Esteve-Codina A, Ruiz-Herrera A, Ibáñez E, Santaló J. The pluripotency state of human embryonic stem cells derived from single blastomeres of eight-cell embryos. Cells Dev 2024; 179:203935. [PMID: 38914137 DOI: 10.1016/j.cdev.2024.203935] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Revised: 06/17/2024] [Accepted: 06/20/2024] [Indexed: 06/26/2024]
Abstract
Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs.
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Affiliation(s)
- Ot Massafret
- Genome Integrity and Reproductive Biology Group, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; Bioengineering in Reproductive Health, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
| | - Montserrat Barragán
- Basic Research Laboratory, Eugin Group, Parc Científic de Barcelona, 08028 Barcelona, Spain
| | - Lucía Álvarez-González
- Genome Integrity and Reproductive Biology Group, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
| | - Begoña Aran
- Stem Cell Bank, Regenerative Medicine Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), 08908 L'Hospitalet de Llobregat, Spain
| | - Beatriz Martín-Mur
- CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, 08028 Barcelona, Spain
| | - Anna Esteve-Codina
- CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, 08028 Barcelona, Spain.; Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Aurora Ruiz-Herrera
- Genome Integrity and Reproductive Biology Group, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
| | - Elena Ibáñez
- Genome Integrity and Reproductive Biology Group, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
| | - Josep Santaló
- Genome Integrity and Reproductive Biology Group, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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12
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Fan W, Yuan H, Chang L, Li Q, Gao J, Ma L, Chen L, Dai Y, Pan X, Zhu X. Role of the TGF-β signaling pathway in induced pluripotent stem cells reprogramming. Chin Med J (Engl) 2024:00029330-990000000-01157. [PMID: 39039625 PMCID: PMC11407801 DOI: 10.1097/cm9.0000000000003229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2023] [Indexed: 07/24/2024] Open
Affiliation(s)
- Weiwen Fan
- The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, Kunming, Yunnan 650032, China
| | - Heling Yuan
- The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming, Yunnan 650032, China
| | - Le Chang
- The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming, Yunnan 650032, China
| | - Qiang Li
- The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, Kunming, Yunnan 650032, China
| | - Jing Gao
- The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming, Yunnan 650032, China
| | - Lihua Ma
- The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming, Yunnan 650032, China
| | - Lvzhe Chen
- The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, Kunming, Yunnan 650032, China
| | - Ying Dai
- The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming, Yunnan 650032, China
| | - Xinghua Pan
- The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, Kunming, Yunnan 650032, China
- The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming, Yunnan 650032, China
- The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming, Yunnan 650032, China
| | - Xiangqing Zhu
- The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, Kunming, Yunnan 650032, China
- The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming, Yunnan 650032, China
- The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming, Yunnan 650032, China
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13
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Lewis PA, Silajdžić E, Smith H, Bates N, Smith CA, Mancini FE, Knight D, Denning C, Brison DR, Kimber SJ. A secreted proteomic footprint for stem cell pluripotency. PLoS One 2024; 19:e0299365. [PMID: 38875182 PMCID: PMC11178176 DOI: 10.1371/journal.pone.0299365] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Accepted: 02/08/2024] [Indexed: 06/16/2024] Open
Abstract
With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.
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Affiliation(s)
- Philip A. Lewis
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Edina Silajdžić
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Helen Smith
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Nicola Bates
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Christopher A. Smith
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Fabrizio E. Mancini
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - David Knight
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Chris Denning
- Biodiscovery Institute, Division of Cancer & Stem Cells, School of Medicine, University of Nottingham, University Park, Nottingham, United Kingdom
| | - Daniel R. Brison
- Royal Manchester Children’s Hospital, Manchester, United Kingdom
| | - Susan J. Kimber
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
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14
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Peng M, Keppeke GD, Tsai LK, Chang CC, Liu JL, Sung LY. The IMPDH cytoophidium couples metabolism and fetal development in mice. Cell Mol Life Sci 2024; 81:210. [PMID: 38717553 PMCID: PMC11078715 DOI: 10.1007/s00018-024-05233-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Revised: 04/04/2024] [Accepted: 04/05/2024] [Indexed: 05/12/2024]
Abstract
The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.
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Affiliation(s)
- Min Peng
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan
| | - Gerson D Keppeke
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
- Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad Católica del Norte, Coquimbo, Chile
| | - Li-Kuang Tsai
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan
| | - Chia-Chun Chang
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan.
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK.
| | - Li-Ying Sung
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan.
- Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, 106, Taiwan.
- Center for Biotechnology, National Taiwan University, Taipei, 106, Taiwan.
- Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan.
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15
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Feng L, Wang Y, Fu Y, Li T, He G. Stem Cell-Based Strategies: The Future Direction of Bioartificial Liver Development. Stem Cell Rev Rep 2024; 20:601-616. [PMID: 38170319 DOI: 10.1007/s12015-023-10672-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/21/2023] [Indexed: 01/05/2024]
Abstract
Acute liver failure (ALF) results from severe liver damage or end-stage liver disease. It is extremely fatal and causes serious health and economic burdens worldwide. Once ALF occurs, liver transplantation (LT) is the only definitive and recommended treatment; however, LT is limited by the scarcity of liver grafts. Consequently, the clinical use of bioartificial liver (BAL) has been proposed as a treatment strategy for ALF. Human primary hepatocytes are an ideal cell source for these methods. However, their high demand and superior viability prevent their widespread use. Hence, finding alternatives that meet the seed cell quality and quantity requirements is imperative. Stem cells with self-renewing, immunogenic, and differentiative capacities are potential cell sources. MSCs and its secretomes encompass a spectrum of beneficial properties, such as anti-inflammatory, immunomodulatory, anti-ROS (reactive oxygen species), anti-apoptotic, pro-metabolomic, anti-fibrogenesis, and pro-regenerative attributes. This review focused on the recent status and future directions of stem cell-based strategies in BAL for ALF. Additionally, we discussed the opportunities and challenges associated with promoting such strategies for clinical applications.
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Affiliation(s)
- Lei Feng
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, 550000, Guizhou, China.
| | - Yi Wang
- Shanxi Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, 030013, Shanxi, China
| | - Yu Fu
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China
| | - Ting Li
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510140, Guangdong, China.
| | - Guolin He
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
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16
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Ding Y, Zhou G, Hu W. Epigenetic regulation of TGF-β pathway and its role in radiation response. Int J Radiat Biol 2024; 100:834-848. [PMID: 38506660 DOI: 10.1080/09553002.2024.2327395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Accepted: 02/27/2024] [Indexed: 03/21/2024]
Abstract
PURPOSE Transforming growth factor (TGF-β) plays a dual role in tumor progression as well as a pivotal role in radiation response. TGF-β-related epigenetic regulations, including DNA methylation, histone modifications (including methylation, acetylation, phosphorylation, ubiquitination), chromatin remodeling and non-coding RNA regulation, have been found to affect the occurrence and development of tumors as well as their radiation response in multiple dimensions. Due to the significance of radiotherapy in tumor treatment and the essential roles of TGF-β signaling in radiation response, it is important to better understand the role of epigenetic regulation mechanisms mediated by TGF-β signaling pathways in radiation-induced targeted and non-targeted effects. CONCLUSIONS By revealing the epigenetic mechanism related to TGF-β-mediated radiation response, summarizing the existing relevant adjuvant strategies for radiotherapy based on TGF-β signaling, and discovering potential therapeutic targets, we hope to provide a new perspective for improving clinical treatment.
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Affiliation(s)
- Yunan Ding
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China
| | - Guangming Zhou
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China
| | - Wentao Hu
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China
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17
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Cheng YS, Taniguchi Y, Yunoki Y, Masai S, Nogi M, Doi H, Sekiguchi K, Nakagawa M. Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells. Regen Ther 2024; 25:113-127. [PMID: 38226057 PMCID: PMC10788407 DOI: 10.1016/j.reth.2023.12.008] [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: 10/03/2023] [Revised: 12/07/2023] [Accepted: 12/17/2023] [Indexed: 01/17/2024] Open
Abstract
Introduction Basic fibroblast growth factor (bFGF, FGF2) and integrin α6β1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear. Methods To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB). Result We identified an interaction between bFGF and integrin α6β1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin. Conclusion Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.
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Affiliation(s)
- Yu-Shen Cheng
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Yukimasa Taniguchi
- Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan
| | - Yasuhiro Yunoki
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Satomi Masai
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Mizuho Nogi
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Hatsuki Doi
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Kiyotoshi Sekiguchi
- Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan
| | - Masato Nakagawa
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
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18
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Lan X, Guo L, Hu C, Zhang Q, Deng J, Wang Y, Chen ZJ, Yan J, Li Y. Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype. Cell Commun Signal 2024; 22:61. [PMID: 38263146 PMCID: PMC10807102 DOI: 10.1186/s12964-023-01463-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Accepted: 12/27/2023] [Indexed: 01/25/2024] Open
Abstract
BACKGROUND During human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation. METHODS Primary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy. RESULTS Our results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-β type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos. CONCLUSIONS Our study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.
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Affiliation(s)
- Xiangxin Lan
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Ling Guo
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Cuiping Hu
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Qian Zhang
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Jianye Deng
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Yufeng Wang
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
| | - Zi-Jiang Chen
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China
- Research Unit of Gametogenesis and Health of ART-Offspring Chinese Academy of Medical Sciences (No. 2021RU001), Jinan, 250012, Shandong, China
| | - Junhao Yan
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China.
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China.
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China.
| | - Yan Li
- Institute of Women, Children and Reproductive Health, Shandong University, Jinan, 250012, Shandong, China.
- State Key Laboratory of Reproductive Medicine and Offspring Health, Shandong University, Jinan, 250012, Shandong, China.
- Medical Integration and Practice Center, Shandong University, Jinan, 250012, Shandong, China.
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Şişli HB, Hayal TB, Şenkal S, Bulut E, Kıratlı B, Asutay AB, Şahin F, Bayrak ÖF, Doğan A. Activation of Wnt Pathway Suppresses Growth of MUG-Chor1 Chordoma Cell Line. Cell Biochem Biophys 2023; 81:823-837. [PMID: 37751039 DOI: 10.1007/s12013-023-01178-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/15/2023] [Indexed: 09/27/2023]
Abstract
Chordoma as a malignant bone tumor, occurs along the axial skeleton and does not have an effective therapy. Brachyury, which is a crucial player for the formation of early embryonic notochord, is abundantly found in both sporadic and familial chordoma. During embryonic development, Brachyury expression was reported to be regulated by the Wnt pathway. The objective of the study is to investigate the role of Wnt signaling in a human chordoma cell line in terms of proliferation, survival, and invasiveness. We tried to elucidate the signaling events that regulate Chordoma cancer. In this regard, Wnt pathway was activated or inhibited using various strategies including small molecules, siRNA-based knockdown and overexpression applications. The results indicated the negative regulatory effect of Wnt signaling activity on proliferation and migration capacity of the chordoma cells. It was revealed that when GSK3β was inhibited, the Wnt pathway was activated and negatively regulated T/Bra expression. Activity of the Wnt pathway caused cell cycle arrest, reduced migration potential of the cells, and led to cell death. Therefore, the present study suggests that the Wnt pathway plays a key role in suppressing the proliferation and invasive characteristics of human chordoma cells and has a great potential as a therapeutic target in further clinical studies.
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Affiliation(s)
- Hatice Burcu Şişli
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Taha Bartu Hayal
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Selinay Şenkal
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Ezgi Bulut
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Binnur Kıratlı
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Ayla Burçin Asutay
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Fikrettin Şahin
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey
| | - Ömer Faruk Bayrak
- Department of Medical Genetics, School of Medicine, Yeditepe University, İstanbul, 34755, Turkey
| | - Ayşegül Doğan
- Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey.
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20
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Xu T, Su P, Wu L, Li D, Qin W, Li Q, Zhou J, Miao YL. OCT4 regulates WNT/β-catenin signaling and prevents mesoendoderm differentiation by repressing EOMES in porcine pluripotent stem cells. J Cell Physiol 2023; 238:2855-2866. [PMID: 37942811 DOI: 10.1002/jcp.31135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Revised: 08/29/2023] [Accepted: 09/21/2023] [Indexed: 11/10/2023]
Abstract
The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self-renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/β-catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/β-catenin signaling because the target gene of WNT/β-catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/β-catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription.
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Affiliation(s)
- Tian Xu
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Peng Su
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Linhui Wu
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Delong Li
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Wei Qin
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Qiao Li
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
| | - Jilong Zhou
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
- Frontiers Science Center for Animal Breeding and Sustainable Production (Huazhong Agricultural University), Ministry of Education, Wuhan, China
| | - Yi-Liang Miao
- Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China
- Frontiers Science Center for Animal Breeding and Sustainable Production (Huazhong Agricultural University), Ministry of Education, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
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21
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Krzysiek-Maczka G, Brzozowski T, Ptak-Belowska A. Helicobacter pylori-activated fibroblasts as a silent partner in gastric cancer development. Cancer Metastasis Rev 2023; 42:1219-1256. [PMID: 37460910 PMCID: PMC10713772 DOI: 10.1007/s10555-023-10122-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Accepted: 06/20/2023] [Indexed: 12/18/2023]
Abstract
The discovery of Helicobacter pylori (Hp) infection of gastric mucosa leading to active chronic gastritis, gastroduodenal ulcers, and MALT lymphoma laid the groundwork for understanding of the general relationship between chronic infection, inflammation, and cancer. Nevertheless, this sequence of events is still far from full understanding with new players and mediators being constantly identified. Originally, the Hp virulence factors affecting mainly gastric epithelium were proposed to contribute considerably to gastric inflammation, ulceration, and cancer. Furthermore, it has been shown that Hp possesses the ability to penetrate the mucus layer and directly interact with stroma components including fibroblasts and myofibroblasts. These cells, which are the source of biophysical and biochemical signals providing the proper balance between cell proliferation and differentiation within gastric epithelial stem cell compartment, when exposed to Hp, can convert into cancer-associated fibroblast (CAF) phenotype. The crosstalk between fibroblasts and myofibroblasts with gastric epithelial cells including stem/progenitor cell niche involves several pathways mediated by non-coding RNAs, Wnt, BMP, TGF-β, and Notch signaling ligands. The current review concentrates on the consequences of Hp-induced increase in gastric fibroblast and myofibroblast number, and their activation towards CAFs with the emphasis to the altered communication between mesenchymal and epithelial cell compartment, which may lead to inflammation, epithelial stem cell overproliferation, disturbed differentiation, and gradual gastric cancer development. Thus, Hp-activated fibroblasts may constitute the target for anti-cancer treatment and, importantly, for the pharmacotherapies diminishing their activation particularly at the early stages of Hp infection.
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Affiliation(s)
- Gracjana Krzysiek-Maczka
- Department of Physiology, the Faculty of Medicine, Jagiellonian University Medical College, 16 Grzegorzecka Street, 31-531, Kraków, Poland.
| | - Tomasz Brzozowski
- Department of Physiology, the Faculty of Medicine, Jagiellonian University Medical College, 16 Grzegorzecka Street, 31-531, Kraków, Poland.
| | - Agata Ptak-Belowska
- Department of Physiology, the Faculty of Medicine, Jagiellonian University Medical College, 16 Grzegorzecka Street, 31-531, Kraków, Poland
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22
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Wang X, Song C, Ye Y, Gu Y, Li X, Chen P, Leng D, Xiao J, Wu H, Xie S, Liu W, Zhao Q, Chen D, Chen X, Wu Q, Chen G, Zhang W. BRD9-mediated control of the TGF-β/Activin/Nodal pathway regulates self-renewal and differentiation of human embryonic stem cells and progression of cancer cells. Nucleic Acids Res 2023; 51:11634-11651. [PMID: 37870468 PMCID: PMC10681724 DOI: 10.1093/nar/gkad907] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 09/29/2023] [Accepted: 10/06/2023] [Indexed: 10/24/2023] Open
Abstract
Bromodomain-containing protein 9 (BRD9) is a specific subunit of the non-canonical SWI/SNF (ncBAF) chromatin-remodeling complex, whose function in human embryonic stem cells (hESCs) remains unclear. Here, we demonstrate that impaired BRD9 function reduces the self-renewal capacity of hESCs and alters their differentiation potential. Specifically, BRD9 depletion inhibits meso-endoderm differentiation while promoting neural ectoderm differentiation. Notably, supplementation of NODAL, TGF-β, Activin A or WNT3A rescues the differentiation defects caused by BRD9 loss. Mechanistically, BRD9 forms a complex with BRD4, SMAD2/3, β-CATENIN and P300, which regulates the expression of pluripotency genes and the activity of TGF-β/Nodal/Activin and Wnt signaling pathways. This is achieved by regulating the deposition of H3K27ac on associated genes, thus maintaining and directing hESC differentiation. BRD9-mediated regulation of the TGF-β/Activin/Nodal pathway is also demonstrated in the development of pancreatic and breast cancer cells. In summary, our study highlights the crucial role of BRD9 in the regulation of hESC self-renewal and differentiation, as well as its participation in the progression of pancreatic and breast cancers.
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Affiliation(s)
- Xuepeng Wang
- The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macao SAR 999078, China
| | - Chengcheng Song
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Ying Ye
- Medical College of Soochow University, Suzhou 215123, China
| | - Yashi Gu
- Zhejiang University–University of Edinburgh Institute (ZJE), Zhejiang University School of Medicine, Zhejiang University, Haining 314400, China
| | - Xuemei Li
- Peninsula Cancer Research Center, School of Basic Medical Sciences, Binzhou Medical University, Yantai 264003, China
| | - Peixin Chen
- Medical College of Soochow University, Suzhou 215123, China
| | - Dongliang Leng
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Jing Xiao
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Hao Wu
- Medical College of Soochow University, Suzhou 215123, China
| | - Sisi Xie
- Zhejiang University–University of Edinburgh Institute (ZJE), Zhejiang University School of Medicine, Zhejiang University, Haining 314400, China
| | - Weiwei Liu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Qi Zhao
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Di Chen
- Zhejiang University–University of Edinburgh Institute (ZJE), Zhejiang University School of Medicine, Zhejiang University, Haining 314400, China
| | - Xi Chen
- Department of Biology, Southern University of Science and Technology, Shenzhen 518000, China
| | - Qiang Wu
- The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macao SAR 999078, China
- The Precision Regenerative Medicine Centre, Macau University of Science and Technology, Taipa, Macao SAR 999078, China
| | - Guokai Chen
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR 999078, China
| | - Wensheng Zhang
- Medical College of Soochow University, Suzhou 215123, China
- Peninsula Cancer Research Center, School of Basic Medical Sciences, Binzhou Medical University, Yantai 264003, China
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255049, China
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23
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McNair AJ, Markby GR, Tang Q, MacRae VE, Corcoran BM. TGF-β phospho antibody array identifies altered SMAD2, PI3K/AKT/SMAD, and RAC signaling contribute to the pathogenesis of myxomatous mitral valve disease. Front Vet Sci 2023; 10:1202001. [PMID: 37908840 PMCID: PMC10613673 DOI: 10.3389/fvets.2023.1202001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Accepted: 08/28/2023] [Indexed: 11/02/2023] Open
Abstract
Background TGFβ signaling appears to contribute to the pathogenesis of myxomatous mitral valve disease (MMVD) in both dogs and humans. However, little is known about the extent of the downstream signaling changes that will then affect cell phenotype and function in both species. Objective Identify changes in downstream signals in the TGFβ pathway in canine MMVD and examine the effects of antagonism of one significant signal (SMAD2 was selected). Materials and methods Canine cultures of normal quiescent valve interstitial cells (qVICs) and disease-derived activated myofibroblasts (aVICs) (n = 6) were examined for TGFβ signaling protein expression using a commercial antibody array. Significant changes were confirmed, and additional proteins of interest downstream in the TGFβ signaling pathway and markers of cell phenotype were examined (PRAS40, S6K, elF4E IRS-1, αSMA, and VIM), using protein immunoblotting. RT-PCR examined expression of gene markers of VIC activation (ACTA2, TAGLN, and MYH10; encoding the proteins αSMA, SM22, and Smemb, respectively). Attenuation of pSMAD2 in aVICs was examined using a combination of RNA interference technology (siRNA) and the SMAD7 (antagonizes SMAD2) agonist asiaticoside. Results The antibody array identified significant changes (P < 0.05) in 19 proteins, of which six were phosphorylated (p). There was increased expression of pSMAD2 and pRAC1 and decreased expression of pmTOR, pERK1/2, and pAKT1. Expression of pPRAS40 and pIRS-1 was increased, as was the mTOR downstream transcription factor pS6K, with increased expression of peIF4E in aVICs, indicating negative feedback control of the PI3K/AKT/mTOR pathway. SMAD2 antagonism by siRNA and the SMAD7 agonist asiaticoside decreased detection of pSMAD by at least 50%, significantly decreased expression of the aVIC gene markers ACTA2, TAGLN, and MYH10, and pαSMA, pAKT2, and pERK1, but had no effect on pS6K, pERK2, or pVIM expression in aVICs. SMAD2 antagonism transitioned diseased aVICs to normal qVICs, while maintaining a mesenchymal phenotype (VIM+) while concurrently affecting non-canonical TGFβ signaling. Conclusion MMVD is associated with changes in both the canonical and non-canonical TGFβ signaling pathway. Antagonism of SMAD2 transitions diseased-activated myofibroblasts back to a normal phenotype, providing data that will inform studies on developing novel therapeutics to treat MMVD in dogs and humans.
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Affiliation(s)
- Andrew J. McNair
- The Roslin Institute, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
| | - Greg R. Markby
- The Roslin Institute, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
| | - Qiyu Tang
- The Roslin Institute, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
| | - Vicky E. MacRae
- The Roslin Institute, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
| | - Brendan M. Corcoran
- The Roslin Institute, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
- Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easterbush Veterinary Centre, Roslin, United Kingdom
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Massagué J, Sheppard D. TGF-β signaling in health and disease. Cell 2023; 186:4007-4037. [PMID: 37714133 PMCID: PMC10772989 DOI: 10.1016/j.cell.2023.07.036] [Citation(s) in RCA: 284] [Impact Index Per Article: 142.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 07/21/2023] [Accepted: 07/28/2023] [Indexed: 09/17/2023]
Abstract
The TGF-β regulatory system plays crucial roles in the preservation of organismal integrity. TGF-β signaling controls metazoan embryo development, tissue homeostasis, and injury repair through coordinated effects on cell proliferation, phenotypic plasticity, migration, metabolic adaptation, and immune surveillance of multiple cell types in shared ecosystems. Defects of TGF-β signaling, particularly in epithelial cells, tissue fibroblasts, and immune cells, disrupt immune tolerance, promote inflammation, underlie the pathogenesis of fibrosis and cancer, and contribute to the resistance of these diseases to treatment. Here, we review how TGF-β coordinates multicellular response programs in health and disease and how this knowledge can be leveraged to develop treatments for diseases of the TGF-β system.
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Affiliation(s)
- Joan Massagué
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
| | - Dean Sheppard
- Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA
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25
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Lau HH, Amirruddin NS, Loo LSW, Chan JW, Iich E, Krishnan VG, Hoon S, Teo AKK. FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro. iScience 2023; 26:107265. [PMID: 37502260 PMCID: PMC10368912 DOI: 10.1016/j.isci.2023.107265] [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: 08/11/2022] [Revised: 10/17/2022] [Accepted: 06/28/2023] [Indexed: 07/29/2023] Open
Abstract
The differentiation of human pluripotent stem cells into the SOX17+ definitive endoderm (DE) germ layer is important for generating tissues for regenerative medicine. Multiple developmental and stem cell studies have demonstrated that Activin/Nodal signaling is the primary driver of definitive endoderm formation. Here, we uncover that the FGF2-FGFR-ERK1/2 signaling contributes to mesendoderm and SOX17+ DE formation. Without ERK1/2 signaling, the Activin/Nodal signaling is insufficient to drive mesendoderm and DE formation. Besides FGF2-FGFR-mediated signaling, IGF1R signaling possibly contributes to the ERK1/2 signaling for DE formation. We identified a temporal relationship between Activin/Nodal-SMAD2 and FGF2-FGFR-ERK1/2 signaling in which Activin/Nodal-SMAD2 participates in the initiation of mesendoderm and DE specification that is followed by increasing activity of FGF2-FGFR-ERK1/2 to facilitate and permit the successful generation of SOX17+ DE. Overall, besides the role of Activin/Nodal signaling for DE formation, our findings shed light on the contribution of ERK1/2 signaling for mesendoderm and DE formation.
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Affiliation(s)
- Hwee Hui Lau
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Nur Shabrina Amirruddin
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Larry Sai Weng Loo
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Jun Wei Chan
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
| | - Elhadi Iich
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
| | | | - Shawn Hoon
- Molecular Engineering Lab, IMCB, Proteos, Singapore, Singapore
| | - Adrian Kee Keong Teo
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A∗STAR), Proteos, Singapore, Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
- Precision Medicine Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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Arekatla G, Trenzinger C, Reimann A, Loeffler D, Kull T, Schroeder T. Optogenetic manipulation identifies the roles of ERK and AKT dynamics in controlling mouse embryonic stem cell exit from pluripotency. Dev Cell 2023:S1534-5807(23)00183-1. [PMID: 37207652 DOI: 10.1016/j.devcel.2023.04.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 03/08/2023] [Accepted: 04/14/2023] [Indexed: 05/21/2023]
Abstract
ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.
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Affiliation(s)
- Geethika Arekatla
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
| | - Christoph Trenzinger
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
| | - Andreas Reimann
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
| | - Dirk Loeffler
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
| | - Tobias Kull
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
| | - Timm Schroeder
- Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland.
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27
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Yoo DH, Im YS, Oh JY, Gil D, Kim YO. DUSP6 is a memory retention feedback regulator of ERK signaling for cellular resilience of human pluripotent stem cells in response to dissociation. Sci Rep 2023; 13:5683. [PMID: 37029196 PMCID: PMC10082014 DOI: 10.1038/s41598-023-32567-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Accepted: 03/29/2023] [Indexed: 04/09/2023] Open
Abstract
Cultured human pluripotent stem cells (hPSCs) grow as colonies that require breakdown into small clumps for further propagation. Although cell death mechanism by single-cell dissociation of hPSCs has been well defined, how hPSCs respond to the deadly stimulus and recover the original status remains unclear. Here we show that dissociation of hPSCs immediately activates ERK, which subsequently activates RSK and induces DUSP6, an ERK-specific phosphatase. Although the activation is transient, DUSP6 expression persists days after passaging. DUSP6 depletion using the CRISPR/Cas9 system reveals that DUSP6 suppresses the ERK activity over the long term. Elevated ERK activity by DUSP6 depletion increases both viability of hPSCs after single-cell dissociation and differentiation propensity towards mesoderm and endoderm lineages. These findings provide new insights into how hPSCs respond to dissociation in order to maintain pluripotency.
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Affiliation(s)
- Dae Hoon Yoo
- Division of Intractable Disease Research, Korea National Institute of Health, Osong, Cheongju, 28160, Republic of Korea
| | - Young Sam Im
- Division of Intractable Disease Research, Korea National Institute of Health, Osong, Cheongju, 28160, Republic of Korea
| | - Ji Young Oh
- Division of Intractable Disease Research, Korea National Institute of Health, Osong, Cheongju, 28160, Republic of Korea
| | - Dayeon Gil
- Division of Intractable Disease Research, Korea National Institute of Health, Osong, Cheongju, 28160, Republic of Korea
| | - Yong-Ou Kim
- Division of Intractable Disease Research, Korea National Institute of Health, Osong, Cheongju, 28160, Republic of Korea.
- Center for National Stem Cell and Regenerative Medicine 202, Osongsaengmyung 2-Ro, Heundeok-Gu, Cheongju, Chungcheongbuk-Do, 28160, Republic of Korea.
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28
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Madrigal P, Deng S, Feng Y, Militi S, Goh KJ, Nibhani R, Grandy R, Osnato A, Ortmann D, Brown S, Pauklin S. Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation. Nat Commun 2023; 14:405. [PMID: 36697417 PMCID: PMC9876972 DOI: 10.1038/s41467-023-36116-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 01/17/2023] [Indexed: 01/26/2023] Open
Abstract
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
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Affiliation(s)
- Pedro Madrigal
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, UK
- Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 0SZ, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, UK
| | - Siwei Deng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Yuliang Feng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Kim Jee Goh
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- The Francis Crick Institute, London, NW1 1AT, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Rodrigo Grandy
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Anna Osnato
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Daniel Ortmann
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Stephanie Brown
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK.
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Legier T, Rattier D, Llewellyn J, Vannier T, Sorre B, Maina F, Dono R. Epithelial disruption drives mesendoderm differentiation in human pluripotent stem cells by enabling TGF-β protein sensing. Nat Commun 2023; 14:349. [PMID: 36681697 PMCID: PMC9867713 DOI: 10.1038/s41467-023-35965-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Accepted: 01/10/2023] [Indexed: 01/22/2023] Open
Abstract
The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.
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Affiliation(s)
- Thomas Legier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Diane Rattier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Jack Llewellyn
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Thomas Vannier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Benoit Sorre
- Institut Curie, Universite ́PSL, Sorbonne Universite ́, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
| | - Flavio Maina
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Rosanna Dono
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France.
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Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers. Theriogenology 2023; 196:37-49. [PMID: 36379144 DOI: 10.1016/j.theriogenology.2022.10.042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Revised: 10/28/2022] [Accepted: 10/31/2022] [Indexed: 11/08/2022]
Abstract
A suitable microenvironment or niche is essential for self-renewal and pluripotency of stem cells cultured in vitro, including bovine embryonic stem cells (bESCs). Feeder cells participate in the construction of stem cell niche by secreting growth factors and extracellular matrix proteins. In this study, metabolomics and transcriptomics analyses were used to investigate the effects of low-density feeder cells on bESCs. The results showed that bESCs co-cultured with low-density feeder cells experienced a decrease in pluripotent gene expression, cell differentiation, and a reduction of central carbon metabolic activity. When cell-permeable pyruvate (Pyr) and recombinant human basic fibroblast growth factor (rhbFGF) were added to the culture system, the pluripotency of bESCs on low-density feeder layers was rescued, and acetyl-coenzyme A (AcCoA) synthesis and fatty acid de novo synthesis increased. In addition, rhbFGF enhances the effects of Pyr and activates the overall metabolic level of bESCs grown on low-density feeder layers. This study explored the rescue effects of exogenous Pyr and rhbFGF on bESCs cultured on low-density feeder layers, which will provide a reference for improvement of the PSC culture system through the supplementation of energy metabolites and growth factors.
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31
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Furlan G, Huyghe A, Combémorel N, Lavial F. Molecular versatility during pluripotency progression. Nat Commun 2023; 14:68. [PMID: 36604434 PMCID: PMC9814743 DOI: 10.1038/s41467-022-35775-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Accepted: 12/22/2022] [Indexed: 01/07/2023] Open
Abstract
A challenge during development is to ensure lineage segregation while preserving plasticity. Using pluripotency progression as a paradigm, we review how developmental transitions are coordinated by redeployments, rather than global resettings, of cellular components. We highlight how changes in response to extrinsic cues (FGF, WNT, Activin/Nodal, Netrin-1), context- and stoichiometry-dependent action of transcription factors (Oct4, Nanog) and reconfigurations of epigenetic regulators (enhancers, promoters, TrxG, PRC) may confer robustness to naïve to primed pluripotency transition. We propose the notion of Molecular Versatility to regroup mechanisms by which molecules are repurposed to exert different, sometimes opposite, functions in close stem cell configurations.
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Affiliation(s)
- Giacomo Furlan
- Cellular reprogramming, stem cells and oncogenesis laboratory - Equipe labellisée La Ligue Contre le Cancer - LabEx Dev2Can - Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, Lyon, 69008, France
- Lunenfeld-Tanenbaum Research Institute, University of Toronto, Toronto, ON, Canada
| | - Aurélia Huyghe
- Cellular reprogramming, stem cells and oncogenesis laboratory - Equipe labellisée La Ligue Contre le Cancer - LabEx Dev2Can - Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, Lyon, 69008, France
| | - Noémie Combémorel
- Cellular reprogramming, stem cells and oncogenesis laboratory - Equipe labellisée La Ligue Contre le Cancer - LabEx Dev2Can - Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, Lyon, 69008, France
| | - Fabrice Lavial
- Cellular reprogramming, stem cells and oncogenesis laboratory - Equipe labellisée La Ligue Contre le Cancer - LabEx Dev2Can - Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, Lyon, 69008, France.
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32
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Chen Y, Lu C, Shang X, Wu K, Chen K. Primary cilia: The central role in the electromagnetic field induced bone healing. Front Pharmacol 2022; 13:1062119. [DOI: 10.3389/fphar.2022.1062119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2022] [Accepted: 11/07/2022] [Indexed: 12/03/2022] Open
Abstract
Primary cilia have emerged as the cellular “antenna” that can receive and transduce extracellular chemical/physical signals, thus playing an important role in regulating cellular activities. Although the electromagnetic field (EMF) is an effective treatment for bone fractures since 1978, however, the detailed mechanisms leading to such positive effects are still unclear. Primary cilia may play a central role in receiving EMF signals, translating physical signals into biochemical information, and initiating various signalingsignaling pathways to transduce signals into the nucleus. In this review, we elucidated the process of bone healing, the structure, and function of primary cilia, as well as the application and mechanism of EMF in treating fracture healing. To comprehensively understand the process of bone healing, we used bioinformatics to analyze the molecular change and associated the results with other studies. Moreover, this review summarizedsummarized some limitations in EMFs-related research and provides an outlook for ongoing studies. In conclusion, this review illustrated the primary cilia and related molecular mechanisms in the EMF-induced bone healing process, and it may shed light on future research.
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33
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Yang XC, Wu XL, Li WH, Wu XJ, Shen QY, Li YX, Peng S, Hua JL. OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation. Zool Res 2022; 43:911-922. [PMID: 36052561 PMCID: PMC9700490 DOI: 10.24272/j.issn.2095-8137.2022.220] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Accepted: 09/01/2022] [Indexed: 08/18/2023] Open
Abstract
As a transcription factor of the Pit-Oct-Unc (POU) domain family, octamer-binding transcription factor 6 ( OCT6) participates in various aspects of stem cell development and differentiation. At present, however, its role in porcine-induced pluripotent stem cells (piPSCs) remains unclear. Here, we explored the function of OCT6 in piPSCs. We found that piPSCs overexpressing OCT6 maintained colony morphology and pluripotency under differentiation conditions, with a similar gene expression pattern to that of non-differentiated piPSCs. Functional analysis revealed that OCT6 attenuated the adverse effects of extracellular signal-regulated kinase (ERK) signaling pathway inhibition on piPSC pluripotency by activating phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling activity. Our research sheds new light on the mechanism by which OCT6 promotes PSC maintenance.
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Affiliation(s)
- Xin-Chun Yang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Xiao-Long Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Wen-Hao Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Xiao-Jie Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Qiao-Yan Shen
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Yun-Xiang Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Sha Peng
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Jin-Lian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China. E-mail:
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34
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Yoney A, Bai L, Brivanlou AH, Siggia ED. Mechanisms underlying WNT-mediated priming of human embryonic stem cells. Development 2022; 149:dev200335. [PMID: 35815787 PMCID: PMC9357376 DOI: 10.1242/dev.200335] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Accepted: 06/23/2022] [Indexed: 11/10/2023]
Abstract
Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.
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Affiliation(s)
- Anna Yoney
- Center for Studies in Physics and Biology, The Rockefeller University, New York, NY 10065, USA
- Laboratory of Synthetic Embryology, The Rockefeller University, New York, NY 10065, USA
| | - Lu Bai
- Department of Biochemistry and Molecular Biology, Department of Physics, Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA 16802, USA
| | - Ali H. Brivanlou
- Laboratory of Synthetic Embryology, The Rockefeller University, New York, NY 10065, USA
| | - Eric D. Siggia
- Center for Studies in Physics and Biology, The Rockefeller University, New York, NY 10065, USA
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35
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Mtshali Z, Govender N, Naicker T. Circulating levels of transforming growth factor beta-1, 2 and 3 in HIV associated preeclamptic pregnancies. J OBSTET GYNAECOL 2022; 42:2853-2859. [PMID: 36006052 DOI: 10.1080/01443615.2022.2110458] [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: 01/11/2023]
Abstract
The active role of transforming growth factor-beta in implantation, embryonic development and decidualization has driven our interest to evaluate circulating TGF-β(1-3) in the synergy of HIV associated pregnancy. Serum TGF-β(1-3) was quantified in normotensive (n = 38) and preeclamptic (n = 38) pregnant women, who were stratified by HIV status, HIV negative (n = 19) and HIV positive (n = 19), using a Bioplex immunoassay.Based on HIV status, we report no significant difference in TGF-β-1 (p = .95) and TGF-β2 (p = .80) however, TGF-β3 was significantly downregulated in HIV positive (p = .03) vs the HIV negative groups. A significant positive correlation (p < .05) was noted between TGF-β3 and gestational age (p = .03) (r = 0.51), birth weight (p = .04) (r = 0.53) and CD4 count (p = .02) (r = 0.53). Bivariate correlation between isoforms based on HIV status showed several significant positive associations. In the synergy of HIV infected PE, we demonstrate an association between TGF-β(1-3) with PE emanating from the hypoxic microenvironment that affects receptor-SMAD activity. Decreased TGF-β3 levels in HIV infected PE, may originate from ARV usage and/or the mutational/physiological dysregulation of SMAD expression. Impact StatementWhat is already known on this subject? TGF-β overexpression can convert its protective functions into pathogenic variants. It has a significant role in the oxidatively stressed and inflammatory condition of tissue fibrosis and hence may also be dysregulated in the microenvironment of PE. In HIV infection, TGF-β promotes viral replication and spreading through the induction of cellular proteins which induce TGF-β production. Also, mononuclear phagocytes infected with HIV also produce increased TGF-β mRNA and proteins.What do the results of the study add? Our results show no association of TGF-β isoforms (1-3) based on pregnancy type (PE vs normotensive pregnant) at term. The lack of association may be linked to TGF-βs dual promoter/suppresser nature or to gestational age.What are the implications of these findings for clinical practice and/or further research? Large-scale comprehensive clinical trials are warranted to elucidate the association and mechanistic role of TGF-β receptor-SMAD signalling, the effect of its inhibitors on cell invasion and angiogenesis as well as to deliver valuable data for the detection of novel therapeutic agents in pregnancies complicated by HIV infection.
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Affiliation(s)
- Zamahlabangane Mtshali
- Discipline of Optics and Imaging, Doris Duke Medical Research Institute, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa
| | - Nalini Govender
- Department of Basic Medical Sciences, Durban University of Technology, Durban, South Africa
| | - Thajasvarie Naicker
- Discipline of Optics and Imaging, Doris Duke Medical Research Institute, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa
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36
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Yadav S, Garrido A, Hernández MC, Oliveros JC, Pérez-García V, Fraga MF, Carrera AC. PI3Kβ-regulated β-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression. Stem Cell Reports 2022; 17:2239-2255. [PMID: 36179694 PMCID: PMC9561645 DOI: 10.1016/j.stemcr.2022.09.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Revised: 09/01/2022] [Accepted: 09/02/2022] [Indexed: 11/21/2022] Open
Abstract
The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kβ) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kβ non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear β-catenin accumulation. β-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kβ epigenetic control of EZH2/β-catenin might be modulated to direct stem cell differentiation.
PI3Kβ directs epigenetic control of stemness and primitive streak (PS) essential genes PI3Kβ directs RAC1/JNK/β-catenin activation and induces EZH2 promoter displacement β-Catenin/EZH2 control NODAL, a gene essential for stemness and the master PS genes PI3Kβ/PI3K activities cooperate at stemness; PI3Kβ directs PS gene expression
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Affiliation(s)
- Sudhanshu Yadav
- Department of Immunology and Oncology, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Antonio Garrido
- Department of Immunology and Oncology, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - M Carmen Hernández
- Department of Immunology and Oncology, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Juan C Oliveros
- Department of Systems Biology, Bioinformatics, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Vicente Pérez-García
- Centro de Investigación Príncipe Felipe, Eduardo Primo Yúfera, 46013 Valencia, Spain
| | - Mario F Fraga
- Nanomaterials and Nanotechnology Research Center/CSIC, Health Research Institute of Asturias (ISPA), Institute of Oncology of Asturias (IUOPA), Research Center for Rare Diseases (CIBERER), 33011 Oviedo, Asturias, Spain
| | - Ana C Carrera
- Department of Immunology and Oncology, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
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37
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Liu J, Yuan Z, Wang Q. Pluripotent Stem Cell-derived Strategies to Treat Acute Liver Failure: Current Status and Future Directions. J Clin Transl Hepatol 2022; 10:692-699. [PMID: 36062278 PMCID: PMC9396313 DOI: 10.14218/jcth.2021.00353] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Revised: 01/17/2022] [Accepted: 02/12/2022] [Indexed: 12/04/2022] Open
Abstract
Liver disease has long been a heavy health and economic burden worldwide. Once the disease is out of control and progresses to end-stage or acute organ failure, orthotopic liver transplantation (OLT) is the only therapeutic alternative, and it requires appropriate donors and aggressive administration of immunosuppressive drugs. Therefore, hepatocyte transplantation (HT) and bioartificial livers (BALs) have been proposed as effective treatments for acute liver failure (ALF) in clinics. Although human primary hepatocytes (PHs) are an ideal cell source to support these methods, the large demand and superior viability of PH is needed, which restrains its wide usage. Thus, a finding alternative to meet the quantity and quality of hepatocytes is urgent. In this context, human pluripotent stem cells (PSC), which have unlimited proliferative and differential potential, derived hepatocytes are a promising renewable cell source. Recent studies of the differentiation of PSC into hepatocytes has provided evidence that supports their clinical application. In this review, we discuss the recent status and future directions of the potential use of PSC-derived hepatocytes in treating ALF. We also discuss opportunities and challenges of how to promote such strategies in the common applications in clinical treatments.
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Affiliation(s)
- Jingfeng Liu
- Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen, Guangdong, China
- Shenzhen Key Laboratory of Immunity and Inflammatory Diseases, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
- Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
| | - Zhiming Yuan
- Department of Gastroenterology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
| | - Qingwen Wang
- Shenzhen Key Laboratory of Immunity and Inflammatory Diseases, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
- Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
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TMAO Suppresses Megalin Expression and Albumin Uptake in Human Proximal Tubular Cells Via PI3K and ERK Signaling. Int J Mol Sci 2022; 23:ijms23168856. [PMID: 36012119 PMCID: PMC9407713 DOI: 10.3390/ijms23168856] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 07/22/2022] [Accepted: 07/23/2022] [Indexed: 11/23/2022] Open
Abstract
Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.
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Cheng F, Li M, Thorne RF, Liu G, Yuwei Z, Wu M, Liu L. P21-activated kinase 4 Pak4 maintains embryonic stem cell pluripotency via Akt activation. Stem Cells 2022; 40:892-905. [PMID: 35896382 PMCID: PMC9585903 DOI: 10.1093/stmcls/sxac050] [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: 01/05/2022] [Accepted: 06/27/2022] [Indexed: 11/30/2022]
Abstract
Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3β signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.
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Affiliation(s)
- Fangyuan Cheng
- Division of Life Sciences and Medicine, the first affiliated hospital of University of Science & Technology of China, and CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network. Hefei, Anhui, China
| | - Mingyue Li
- Division of Life Sciences and Medicine, the first affiliated hospital of University of Science & Technology of China, and CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network. Hefei, Anhui, China
| | - Rick Francis Thorne
- Translational Research Institute, Henan Provincial People's Hospital, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan, China.,Henan key Laboratory of Stem cell Differentiation and Modification, Henan Provincial People's Hospital, Henan University, Zhengzhou, Henan, China
| | - Guangzhi Liu
- Henan key Laboratory of Stem cell Differentiation and Modification, Henan Provincial People's Hospital, Henan University, Zhengzhou, Henan, China
| | - Zhang Yuwei
- Henan key Laboratory of Stem cell Differentiation and Modification, Henan Provincial People's Hospital, Henan University, Zhengzhou, Henan, China
| | - Mian Wu
- Division of Life Sciences and Medicine, the first affiliated hospital of University of Science & Technology of China, and CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network. Hefei, Anhui, China.,Translational Research Institute, Henan Provincial People's Hospital, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan, China.,Henan key Laboratory of Stem cell Differentiation and Modification, Henan Provincial People's Hospital, Henan University, Zhengzhou, Henan, China
| | - Lianxin Liu
- Division of Life Sciences and Medicine, the first affiliated hospital of University of Science & Technology of China, and CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network. Hefei, Anhui, China
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40
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Kanke T, Fujii W, Naito K, Sugiura K. Effect of fibroblast growth factor signaling on cumulus expansion in mice in vitro. Mol Reprod Dev 2022; 89:281-289. [PMID: 35678749 DOI: 10.1002/mrd.23616] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 04/23/2022] [Accepted: 05/25/2022] [Indexed: 11/07/2022]
Abstract
The expansion of cumulus cells associated with oocytes is an essential phenomenon in normal mammalian ovulation. Indeed, attenuated expression of cumulus expansion-related genes, including Has2, Ptgs2, Ptx3, and Tnfaip6, results in ovulation failure, leading to female subfertility or infertility. Moreover, emerging evidence suggests that proteins of the fibroblast growth factor (FGF) family, produced within ovarian follicles, regulate the development and function of cumulus cells; however, the effects of FGF signaling on cumulus expansion have not been investigated extensively. Herein, we investigate the effects of FGF signaling, particularly those of FGF8 secreted by oocytes, on epidermal growth factor-induced cumulus expansion in mice. The phosphorylation level of MAPK3/1, an intracellular mediator of FGF signaling, was significantly decreased in cumulus-oocyte complexes (COCs) following treatment with NVP-BGJ398, an FGF receptor inhibitor. Moreover, even though NVP-BGJ398 treatment did not affect cumulus cell expansion, it significantly upregulated the expression of Ptgs2 and Ptx3. In contrast, treatment with recombinant FGF8 did not affect the degree of cumulus expansion or the expression of expansion-related genes in COCs or oocytectomized cumulus cell complexes. Collectively, these results suggest that FGFs, other than FGF8, exert suppressive effects on the cumulus expansion process in mice.
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Affiliation(s)
- Takuya Kanke
- Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Wataru Fujii
- Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Kunihiko Naito
- Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Koji Sugiura
- Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
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Närvä E, Taskinen ME, Lilla S, Isomursu A, Pietilä M, Weltner J, Isola J, Sihto H, Joensuu H, Zanivan S, Norman J, Ivaska J. MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels. iScience 2022; 25:104459. [PMID: 35677646 PMCID: PMC9167974 DOI: 10.1016/j.isci.2022.104459] [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: 10/04/2021] [Revised: 03/04/2022] [Accepted: 05/13/2022] [Indexed: 11/01/2022] Open
Abstract
MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with β3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-β receptor II (TGFBR2), a key modulator of TGF-β signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.
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Affiliation(s)
- Elisa Närvä
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland
| | - Maria E. Taskinen
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland
| | | | - Aleksi Isomursu
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland
| | - Mika Pietilä
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland
| | - Jere Weltner
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland
| | - Jorma Isola
- Laboratory of Cancer Biology, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
| | - Harri Sihto
- Department of Pathology, University of Helsinki, 00290 Helsinki, Finland
| | - Heikki Joensuu
- University of Helsinki and Comprehensive Cancer Center, Helsinki University Hospital, 00290 Helsinki, Finland
| | - Sara Zanivan
- CRUK Beatson Institute, Glasgow G61 1BD, UK
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK
| | - Jim Norman
- CRUK Beatson Institute, Glasgow G61 1BD, UK
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK
| | - Johanna Ivaska
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland
- InFLAMES Research Flagship Center, University of Turku, 20520 Turku, Finland
- Department of Life Technologies, University of Turku, 20520 Turku, Finland
- Western Finnish Cancer Center (FICAN West), University of Turku, 20520 Turku, Finland
- Foundation for the Finnish Cancer Institute, Tukholmankatu 8, Helsinki, Finland
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Jiang H, Du M, Li Y, Zhou T, Lei J, Liang H, Zhong Z, Al-Lamki RS, Jiang M, Yang J. ID proteins promote the survival and primed-to-naive transition of human embryonic stem cells through TCF3-mediated transcription. Cell Death Dis 2022; 13:549. [PMID: 35701409 PMCID: PMC9198052 DOI: 10.1038/s41419-022-04958-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 05/05/2022] [Accepted: 05/16/2022] [Indexed: 01/21/2023]
Abstract
Inhibition of DNA binding proteins 1 and 3 (ID1 and ID3) are important downstream targets of BMP signalling that are necessary for embryonic development. However, their specific roles in regulating the pluripotency of human embryonic stem cells (hESCs) remain unclear. Here, we examined the roles of ID1 and ID3 in primed and naive-like hESCs and showed that ID1 and ID3 knockout lines (IDs KO) exhibited decreased survival in both primed and naive-like state. IDs KO lines in the primed state also tended to undergo pluripotent dissolution and ectodermal differentiation. IDs KO impeded the primed-to-naive transition (PNT) of hESCs, and overexpression of ID1 in primed hESCs promoted PNT. Furthermore, single-cell RNA sequencing demonstrated that ID1 and ID3 regulated the survival and pluripotency of hESCs through the AKT signalling pathway. Finally, we showed that TCF3 mediated transcriptional inhibition of MCL1 promotes AKT phosphorylation, which was confirmed by TCF3 knockdown in KO lines. Our study suggests that IDs/TCF3 acts through AKT signalling to promote survival and maintain pluripotency of both primed and naive-like hESCs.
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Affiliation(s)
- Haibin Jiang
- grid.506261.60000 0001 0706 7839Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China ,grid.13402.340000 0004 1759 700XDepartment of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Mingxia Du
- grid.506261.60000 0001 0706 7839Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China
| | - Yaning Li
- grid.13402.340000 0004 1759 700XDepartment of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Tengfei Zhou
- grid.414906.e0000 0004 1808 0918Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang China
| | - Jia Lei
- grid.13402.340000 0004 1759 700XDepartment of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Hongqing Liang
- grid.13402.340000 0004 1759 700XDivision of Human Reproduction and Developmental Genetics, Women’s Hospital and Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang China
| | - Zhen Zhong
- grid.13402.340000 0004 1759 700XDepartment of human anatomy and histoembryology, Zhejiang University School of Medicine, Hangzhou, Zhejiang China
| | - Rafia S. Al-Lamki
- grid.5335.00000000121885934Department of Medicine, National Institute of Health Research Cambridge Biomedical Research Centre, University of Cambridge, Cambridge, UK
| | - Ming Jiang
- grid.13402.340000 0004 1759 700XDepartment of Gastroenterology of The Children’s Hospital, Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang China
| | - Jun Yang
- grid.13402.340000 0004 1759 700XDepartment of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Pan T, Wang N, Zhang J, Yang F, Chen Y, Zhuang Y, Xu Y, Fang J, You K, Lin X, Li Y, Li S, Liang K, Li YX, Gao Y. Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy. Stem Cell Res Ther 2022; 13:159. [PMID: 35410439 PMCID: PMC8996222 DOI: 10.1186/s13287-022-02831-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Accepted: 03/20/2022] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Various methods have been developed to generate hepatic cells from human pluripotent stem cells (hPSCs) that rely on the combined use of multiple expensive growth factors, limiting industrial-scale production and widespread applications. Small molecules offer an attractive alternative to growth factors for producing hepatic cells since they are more economical and relatively stable. METHODS We dissect small-molecule combinations and identify the ideal cocktails to achieve an optimally efficient and cost-effective strategy for hepatic cells differentiation, expansion, and maturation. RESULTS We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling. Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification. The obtained hepatoblasts (HBs) could subsequently expand and mature into functional hepatocyte-like cells (HLCs) by the established chemical formulas. Thus, we established a stepwise strategy with complete small molecules for efficiently producing scalable HBs and functionally matured HLCs. The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo. CONCLUSION Our current small-molecule-based hepatic generation protocol presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery using.
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Affiliation(s)
- Tingcai Pan
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China.,Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Ning Wang
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Jiaye Zhang
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Fan Yang
- Guangdong Key Laboratory of Non-Human Primate Models, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong, China
| | - Yan Chen
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yuanqi Zhuang
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yingying Xu
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Ji Fang
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Kai You
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Xianhua Lin
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yang Li
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China
| | - Shao Li
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China
| | - Kangyan Liang
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China
| | - Yin-Xiong Li
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
| | - Yi Gao
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China. .,State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China.
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44
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Graffmann N, Scherer B, Adjaye J. In vitro differentiation of pluripotent stem cells into hepatocyte like cells - basic principles and current progress. Stem Cell Res 2022; 61:102763. [DOI: 10.1016/j.scr.2022.102763] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 03/08/2022] [Accepted: 03/22/2022] [Indexed: 12/11/2022] Open
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45
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Metabolic and epigenetic regulation of endoderm differentiation. Trends Cell Biol 2022; 32:151-164. [PMID: 34607773 PMCID: PMC8760149 DOI: 10.1016/j.tcb.2021.09.002] [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] [Received: 07/15/2021] [Revised: 08/31/2021] [Accepted: 09/10/2021] [Indexed: 02/06/2023]
Abstract
The endoderm, one of the three primary germ layers, gives rise to lung, liver, stomach, intestine, colon, pancreas, bladder, and thyroid. These endoderm-originated organs are subject to many life-threatening diseases. However, primary cells/tissues from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells (hPSCs), therefore, hold great promise for generating endodermal cells and their derivatives for the development of new therapeutics against these human diseases. Although a wealth of research has provided crucial information on the mechanisms underlying endoderm differentiation from hPSCs, increasing evidence has shown that metabolism, in connection with epigenetics, actively regulates endoderm differentiation in addition to the conventional endoderm inducing signals. Here we review recent advances in metabolic and epigenetic regulation of endoderm differentiation.
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46
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Su TR, Yu CC, Chao SC, Huang CC, Liao YW, Hsieh PL, Yu CH, Lin SS. Fenofibrate diminishes the self-renewal and metastasis potentials of oral carcinoma stem cells through NF-κB signaling. J Formos Med Assoc 2022; 121:1900-1907. [DOI: 10.1016/j.jfma.2022.01.014] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 01/11/2022] [Accepted: 01/13/2022] [Indexed: 12/24/2022] Open
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Cheng W, Liu F, Ren Z, Chen W, Chen Y, Liu T, Ma Y, Cao N, Wang J. Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens. Nat Commun 2022; 13:478. [PMID: 35078991 PMCID: PMC8789821 DOI: 10.1038/s41467-022-28106-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Accepted: 12/14/2021] [Indexed: 12/12/2022] Open
Abstract
AbstractN6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.
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Pour M, Kumar AS, Farag N, Bolondi A, Kretzmer H, Walther M, Wittler L, Meissner A, Nachman I. Emergence and patterning dynamics of mouse-definitive endoderm. iScience 2022; 25:103556. [PMID: 34988400 PMCID: PMC8693470 DOI: 10.1016/j.isci.2021.103556] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 10/21/2021] [Accepted: 12/01/2021] [Indexed: 11/30/2022] Open
Abstract
The segregation of definitive endoderm (DE) from bipotent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatiotemporal window in the embryo. Here, we employ a dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in vivo in isolated cells within a temporally restricted window. In 2D and 3D in vitro models, DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17 + cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with upregulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.
Sox17 onsets in a few isolated cells within Bra-expressing population Sox17 onset followed by expansion and self-sorting Final number of Sox17+ cells does not depend on self-sorting or cell movement The DE segregation pattern is similar in in vivo and in 2D, 3D in vitro systems
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Affiliation(s)
- Maayan Pour
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Abhishek Sampath Kumar
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Naama Farag
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Adriano Bolondi
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Helene Kretzmer
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Maria Walther
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Lars Wittler
- Department of Developmental Genetics, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Alexander Meissner
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.,Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.,Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Iftach Nachman
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
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Lea RA, McCarthy A, Boeing S, Fallesen T, Elder K, Snell P, Christie L, Adkins S, Shaikly V, Taranissi M, Niakan KK. KLF17 promotes human naïve pluripotency but is not required for its establishment. Development 2021; 148:272511. [PMID: 34661235 PMCID: PMC8645209 DOI: 10.1242/dev.199378] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 10/11/2021] [Indexed: 12/11/2022]
Abstract
Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions. Summary: Given that KLF17 was shown to be sufficient, but not necessary, to establish naïve pluripotent hESCs, KLF17 might function as a peripheral regulator of human pluripotent stem cells.
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Affiliation(s)
- Rebecca A Lea
- Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Afshan McCarthy
- Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Stefan Boeing
- Bioinformatics and Biostatistics Service, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Todd Fallesen
- Crick Advanced Light Microscopy, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Kay Elder
- Bourn Hall Clinic, Bourn, Cambridge CB23 2TN, UK
| | - Phil Snell
- Bourn Hall Clinic, Bourn, Cambridge CB23 2TN, UK
| | | | - Sarah Adkins
- Assisted Reproduction and Gynaecology Centre, London W1G 6LP, UK
| | - Valerie Shaikly
- Assisted Reproduction and Gynaecology Centre, London W1G 6LP, UK
| | | | - Kathy K Niakan
- Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.,The Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
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Human Induced Pluripotent Stem Cell-Derived Vascular Cells: Recent Progress and Future Directions. J Cardiovasc Dev Dis 2021; 8:jcdd8110148. [PMID: 34821701 PMCID: PMC8622843 DOI: 10.3390/jcdd8110148] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 11/01/2021] [Accepted: 11/02/2021] [Indexed: 12/12/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) hold great promise for cardiovascular regeneration following ischemic injury. Considerable effort has been made toward the development and optimization of methods to differentiate hiPSCs into vascular cells, such as endothelial and smooth muscle cells (ECs and SMCs). In particular, hiPSC-derived ECs have shown robust potential for promoting neovascularization in animal models of cardiovascular diseases, potentially achieving significant and sustained therapeutic benefits. However, the use of hiPSC-derived SMCs that possess high therapeutic relevance is a relatively new area of investigation, still in the earlier investigational stages. In this review, we first discuss different methodologies to derive vascular cells from hiPSCs with a particular emphasis on the role of key developmental signals. Furthermore, we propose a standardized framework for assessing and defining the EC and SMC identity that might be suitable for inducing tissue repair and regeneration. We then highlight the regenerative effects of hiPSC-derived vascular cells on animal models of myocardial infarction and hindlimb ischemia. Finally, we address several obstacles that need to be overcome to fully implement the use of hiPSC-derived vascular cells for clinical application.
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