|
1
|
Chen X, Shi C, Ye Y, Wang C, Li R, Wang H, Hou C, Song W, Xu X, Mu C. Dorsomorphin (DM) inhibits the ovarian development of Portunus trituberculatus by acting on the BMP signaling pathway. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART D, GENOMICS & PROTEOMICS 2025; 54:101440. [PMID: 39954577 DOI: 10.1016/j.cbd.2025.101440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 01/17/2025] [Accepted: 02/07/2025] [Indexed: 02/17/2025]
Abstract
Bone morphogenic proteins (BMPs) regulate animal growth, cell proliferation and differentiation. The BMP signaling pathway plays an important regulatory role during ovarian follicle development in mammals. However, related studies in crustaceans are limited. The focus of this study was the key gene of the BMP signaling pathway, the BMP type I receptor. Portunus trituberculatus was injected with different concentrations of dorsomorphin (DM) and observed for one month to identify the optimal effective concentration for interference with the BMP signaling pathway. Subsequent transcriptomics, proteomics, and metabolomics measurements were performed to identify the effects of BMP signaling on ovarian development in P. trituberculatus. A preliminary mechanism of action of the BMP signaling pathway in the regulation of ovarian development was revealed through combined multiomics analysis and lipid analysis. This study provides a theoretical basis for further exploration of the molecular mechanism regulating gonadal development in crustaceans.
Collapse
Affiliation(s)
- Xiaocong Chen
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang 222005, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Ce Shi
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Yangfang Ye
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Chunlin Wang
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Ronghua Li
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Huan Wang
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Congcong Hou
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Weiwei Song
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China
| | - Xinghong Xu
- Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang 222005, China
| | - Changkao Mu
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo 315211, China.
| |
Collapse
|
|
2
|
Chen X, Shi C, Ye Y, Wang C, Li R, Wang H, Hou C, Song W, Mu C. FK506 activates the BMP signaling pathway to regulate ovarian development in Portunus trituberculatus. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2025; 166:105365. [PMID: 40169079 DOI: 10.1016/j.dci.2025.105365] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/21/2025] [Revised: 03/10/2025] [Accepted: 03/29/2025] [Indexed: 04/03/2025]
Abstract
Bone morphogenic proteins (BMPs) play important regulatory roles in the development of follicles in mammals. However, studies on the roles of BMPs in ovarian development in low-level aquatic animals, especially the swimming crab Portunus trituberculatus, are limited. In this study, a BMP Ⅰ-type receptor-specific activator (tacrolimus, FK506) was administered at different concentrations via in vivo injection, and the effects of FK506 on the regulation of the BMP signaling pathway during ovarian development in P. trituberculatus were examined. The tissue and cell morphology was observed, and a combined transcriptomics, proteomics and metabolomics analysis was carried out. Crabs administered FK506 exhibited elevated GSI alongside reduced HSI compared to control and blank groups. The main biological processes enriched by joint analysis included lipid metabolism, sugar metabolism, and amino acid metabolism. Fatty acid composition analysis revealed that the activator may activate the BMP signaling pathway to promote ovarian development and accelerate the transport of unsaturated fatty acids from the hepatopancreas to the ovaries. Amino acid metabolism and carbohydrate metabolism provide transporter proteins and energy for lipid metabolism. This study is highly important because it reveals the molecular mechanism by which the BMP signaling pathway regulates gonadal development in a crustacean.
Collapse
Affiliation(s)
- Xiaocong Chen
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, 222005, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Ce Shi
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Yangfang Ye
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Chunlin Wang
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Ronghua Li
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Huan Wang
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Congcong Hou
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Weiwei Song
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| | - Changkao Mu
- Key Laboratory of Applied Aquacultral Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, China; Key Laboratory of Green Mariculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Ningbo, 315211, China
| |
Collapse
|
|
3
|
Hua L, Peng Y, Yan L, Yuan P, Qiao J. Moving toward totipotency: the molecular and cellular features of totipotent and naive pluripotent stem cells. Hum Reprod Update 2025:dmaf006. [PMID: 40299455 DOI: 10.1093/humupd/dmaf006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 01/06/2025] [Indexed: 04/30/2025] Open
Abstract
BACKGROUND Dissecting the key molecular mechanism of embryonic development provides novel insights into embryogenesis and potential intervention strategies for clinical practices. However, the ability to study the molecular mechanisms of early embryo development in humans, such as zygotic genome activation and lineage segregation, is meaningfully constrained by methodological limitations and ethical concerns. Totipotent stem cells have an extended developmental potential to differentiate into embryonic and extraembryonic tissues, providing a suitable model for studying early embryo development. Recently, a series of ground-breaking results on stem cells have identified totipotent-like cells or induced pluripotent stem cells into totipotent-like cells. OBJECTIVE AND RATIONALE This review followed the PRISMA guidelines, surveys the current works of literature on totipotent, naive, and formative pluripotent stem cells, introduces the molecular and biological characteristics of those stem cells, and gives advice for future research. SEARCH METHODS The search method employed the terms 'totipotent' OR 'naive pluripotent stem cell' OR 'formative pluripotent stem cell' for unfiltered search on PubMed, Web of Science, and Cochrane Library. Papers included were those with information on totipotent stem cells, naive pluripotent stem cells, or formative pluripotent stem cells until June 2024 and were published in the English language. Articles that have no relevance to stem cells, or totipotent, naive pluripotent, or formative pluripotent cells were excluded. OUTCOMES There were 152 records included in this review. These publications were divided into four groups according to the species of the cells included in the studies: 67 human stem cell studies, 70 mouse stem cell studies, 9 porcine stem cell studies, and 6 cynomolgus stem cell studies. Naive pluripotent stem cell models have been established in other species such as porcine and cynomolgus. Human and mouse totipotent stem cells, e.g. human 8-cell-like cells, human totipotent blastomere-like cells, and mouse 2-cell-like cells, have been successfully established and exhibit high developmental potency for both embryonic and extraembryonic contributions. However, the observed discrepancies between these cells and real embryos in terms of epigenetics and transcription suggest that further research is warranted. Our results systematically reviewed the established methods, molecular characteristics, and developmental potency of different naive, formative pluripotent, and totipotent stem cells. Furthermore, we provide a parallel comparison between animal and human models, and offer recommendations for future applications to advance early embryo research and assisted reproduction technologies. WIDER IMPLICATIONS Totipotent cell models provide a valuable resource to understand the underlying mechanisms of embryo development and forge new paths toward future treatment of infertility and regenerative medicine. However, current in vitro cell models exhibit epigenetic and transcriptional differences from in vivo embryos, and many cell models are unstable across passages, thus imperfectly recapitulating embryonic development. In this regard, standardizing and expanding current research on totipotent stem cell models are essential to enhance our capability to resemble and decipher embryogenesis.
Collapse
Affiliation(s)
- Lingyue Hua
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China
- National Clinical Research Center for Obstetrics and Gynecology (Peking University Third Hospital), Beijing, China
- Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China
- Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Yuyang Peng
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China
- National Clinical Research Center for Obstetrics and Gynecology (Peking University Third Hospital), Beijing, China
- Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China
- Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
- Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
- Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Liying Yan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China
- National Clinical Research Center for Obstetrics and Gynecology (Peking University Third Hospital), Beijing, China
- Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China
- Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Peng Yuan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China
- National Clinical Research Center for Obstetrics and Gynecology (Peking University Third Hospital), Beijing, China
- Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China
- Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Jie Qiao
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China
- National Clinical Research Center for Obstetrics and Gynecology (Peking University Third Hospital), Beijing, China
- Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China
- Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
- Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
- Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
- Beijing Advanced Innovation Center for Genomics, Beijing, China
| |
Collapse
|
|
4
|
Chaugule S, Constantinou CK, John AA, Micha D, Eekhoff M, Gravallese E, Gao G, Shim JH. Comprehensive Review of Osteogenesis Imperfecta: Current Treatments and Future Innovations. Hum Gene Ther 2025; 36:597-617. [PMID: 39932815 PMCID: PMC11971546 DOI: 10.1089/hum.2024.191] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Accepted: 01/08/2025] [Indexed: 02/13/2025] Open
Abstract
Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility due to reduced bone quality, often accompanied by low bone mass, recurrent fractures, hearing loss, skeletal abnormalities, and short stature. Pathogenic variants in over 20 genes lead to clinical and genetic variability in OI, resulting in diverse symptoms and severity. Current management involves a multidisciplinary approach, including antiresorptive medications, physiotherapy, occupational therapy, and orthopedic surgery, which provide symptomatic relief but no cure. Advancements in gene therapy technologies and stem cell therapies offer promising prospects for long-lasting or permanent solutions. This review provides a comprehensive overview of OI's classification, pathogenesis, and current treatment options. It also explores emerging biotechnologies for stem cells and gene-targeted therapies in OI. The potential of these innovative therapies and their clinical implementation challenges are evaluated, focusing on their imminent success in treating bone disorders.
Collapse
Affiliation(s)
- Sachin Chaugule
- Division of Rheumatology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Department of Genetic and Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
| | | | - Aijaz Ahmad John
- Division of Rheumatology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Department of Genetic and Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
| | - Dimitra Micha
- Department of Human Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam; Amsterdam Rare Bone Disease center, Amsterdam Bone Center, Amsterdam Movement Sciences, Amsterdam Reproduction and Development, Amsterdam, The Netherlands
| | - Marelise Eekhoff
- Department of Internal Medicine, Section Endocrinology & Metabolism, Amsterdam UMC, Vrije Universiteit Amsterdam; Amsterdam Rare Bone Disease center, Amsterdam Bone Center, Amsterdam Movement Sciences, Amsterdam Reproduction and Development Amsterdam, Amsterdam, Netherlands
| | - Ellen Gravallese
- Division of Rheumatology, Inflammation, and Immunity, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA
| | - Guangping Gao
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Viral Vector Core, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
| | - Jae-Hyuck Shim
- Division of Rheumatology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Department of Genetic and Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
- Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
| |
Collapse
|
|
5
|
Parmar B, Bhatia D. Small Molecular Approaches for Cellular Reprogramming and Tissue Engineering: Functions as Mediators of the Cell Signaling Pathway. Biochemistry 2024; 63:2542-2556. [PMID: 39312802 DOI: 10.1021/acs.biochem.4c00427] [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] [Indexed: 09/25/2024]
Abstract
Utilizing induced pluripotent stem cells (iPSCs) in drug screening and cell replacement therapy has emerged as a method with revolutionary applications. With the advent of patient-specific iPSCs and the subsequent development of cells that exhibit disease phenotypes, the focus of medication research will now shift toward the pathology of human diseases. Regular iPSCs can also be utilized to generate cells that assess the negative impacts of medications. These cells provide a much more precise and cost-efficient approach compared to many animal models. In this review, we explore the utilization of small-molecule drugs to enhance the growth of iPSCs and gain insights into the process of reprogramming. We mainly focus on the functions of small molecules in modulating different signaling pathways, thereby modulating cell fate. Understanding the way small molecule drugs interact with iPSC technology has the potential to significantly enhance the understanding of physiological pathways in stem cells and practical applications of iPSC-based therapy and screening systems, revolutionizing the treatment of diseases.
Collapse
Affiliation(s)
- Bhagyesh Parmar
- Department of Biological Sciences and Engineering, Indian Institute of Technology, Palaj, Gandhinagar 382355, India
| | - Dhiraj Bhatia
- Department of Biological Sciences and Engineering, Indian Institute of Technology, Palaj, Gandhinagar 382355, India
| |
Collapse
|
|
6
|
Liu Y, Zhang S, Zou G, An J, Li Y, Lin D, Wang D, Li Y, Chen J, Feng T, Li H, Chen Y, Zhang M, Kumar M, Wang L, Hou R, Liu J. Generation and characterization of giant panda induced pluripotent stem cells. SCIENCE ADVANCES 2024; 10:eadn7724. [PMID: 39303041 DOI: 10.1126/sciadv.adn7724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Accepted: 08/14/2024] [Indexed: 09/22/2024]
Abstract
The giant panda (Ailuropoda melanoleuca) stands as a flagship and umbrella species, symbolizing global biodiversity. While traditional assisted reproductive technology faces constraints in safeguarding the genetic diversity of giant pandas, induced pluripotent stem cells (iPSCs) known for their capacity to differentiate into diverse cells types, including germ cells, present a transformative potential for conservation of endangered animals. In this study, primary fibroblast cells were isolated from the giant panda, and giant panda iPSCs (GPiPSCs) were generated using a non-integrating episomal vector reprogramming method. Characterization of these GPiPSCs revealed their state of primed pluripotency and demonstrated their potential for differentiation. Furthermore, we innovatively formulated a species-specific chemically defined FACL medium and unraveled the intricate signaling pathway networks responsible for maintaining the pluripotency and fostering cell proliferation of GPiPSCs. This study provides key insights into rare species iPSCs, offering materials for panda characteristics research and laying the groundwork for in vitro giant panda gamete generation, potentially aiding endangered species conservation.
Collapse
Affiliation(s)
- Yuliang Liu
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Shihao Zhang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Gaoyang Zou
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Regenerative Medicine and Health Guangdong Laboratory at GIBH, Guangzhou 510530, China
| | - Junhui An
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Yuan Li
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Danni Lin
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Donghui Wang
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Yan Li
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Jiasong Chen
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Tongying Feng
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Hongyan Li
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Yijiao Chen
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Mingyue Zhang
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Manish Kumar
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Luqin Wang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Rong Hou
- Chengdu Research Base of Giant Panda Breeding, Sichuan Province, Chengdu 610081, China
- Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Sichuan Province, Chengdu 610081, China
| | - Jing Liu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Regenerative Medicine and Health Guangdong Laboratory at GIBH, Guangzhou 510530, China
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, P.R.China
| |
Collapse
|
|
7
|
Cai S, Dai Q. Progress in preclinical research on induced pluripotent stem cell therapy for acute myocardial infarction. Zhejiang Da Xue Xue Bao Yi Xue Ban 2024; 53:244-253. [PMID: 38594961 PMCID: PMC11057988 DOI: 10.3724/zdxbyxb-2023-0402] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2023] [Accepted: 03/09/2024] [Indexed: 04/11/2024]
Abstract
Induced pluripotent stem cells (iPSCs) are obtained by introducing exogenous genes or adding chemicals to the culture medium to induce somatic cell differentiation. Similarly to embryonic stem cells, iPSCs have the ability to differentiate into all three embryonic cell lines. iPSCs can differentiate into cardiac muscle cells through two-dimensional differentiation methods such as monolayer cell culture and co-culture, or through embryoid body and scaffold-based three-dimensional differentiation methods. In addition, the process of iPSCs differentiation into cardiac muscle cells also requires activation or inhibition of specific signaling pathways,such as Wnt, BMP, Notch signaling pathways to mimic the development of the heart in vivo. In recent years, suspension culturing in bioreactors has been shown to produce large number of iPSCs derived cardiac muscle cells (iPSC-CMs). Before transplantation, it is necessary to purify iPSC-CMs through metabolic regulation or cell sorting to eliminate undifferentiated iPSCs, which may lead to teratoma formation. The transplantation methods for iPSC-CMs are mainly injection of cell suspension and transplantation of cell patches into the infarcted myocardium. Animal studies have shown that transplantation of iPSC-CMs into the infarcted myocardium can improve cardiac function. This article reviews the progress in preclinical studies on iPSC-CMs therapy for acute myocardial infarction and discusses the limitations and challenges of its clinical application to provide references for further clinical research and application.
Collapse
Affiliation(s)
- Songyan Cai
- Department of Cardiology, First Affiliated Hospital of Kunming Medical University, Kunming 650032, China.
| | - Qingyuan Dai
- Department of Cardiology, First Affiliated Hospital of Kunming Medical University, Kunming 650032, China.
| |
Collapse
|
|
8
|
Oke A, Manohar SM. Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells. Cell Reprogram 2024; 26:46-56. [PMID: 38635924 DOI: 10.1089/cell.2024.0002] [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] [Indexed: 04/20/2024] Open
Abstract
Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.
Collapse
Affiliation(s)
- Anagha Oke
- Department of Biological Sciences, Sunandan Divatia School of Science, NMIMS (Deemed-to-Be) University, Mumbai, Maharashtra, India
| | - Sonal M Manohar
- Department of Biological Sciences, Sunandan Divatia School of Science, NMIMS (Deemed-to-Be) University, Mumbai, Maharashtra, India
| |
Collapse
|
|
9
|
Swaidan NT, Soliman NH, Aboughalia AT, Darwish T, Almeshal RO, Al-Khulaifi AA, Taha RZ, Alanany R, Hussein AY, Salloum-Asfar S, Abdulla SA, Abdallah AM, Emara MM. CCN3, POSTN, and PTHLH as potential key regulators of genomic integrity and cellular survival in iPSCs. Front Mol Biosci 2024; 11:1342011. [PMID: 38375508 PMCID: PMC10875024 DOI: 10.3389/fmolb.2024.1342011] [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: 11/21/2023] [Accepted: 01/11/2024] [Indexed: 02/21/2024] Open
Abstract
Reprogramming human somatic cells into a pluripotent state, achieved through the activation of well-defined transcriptional factors known as OSKM factors, offers significant potential for regenerative medicine. While OSKM factors are a robust reprogramming method, efficiency remains a challenge, with only a fraction of cells undergoing successful reprogramming. To address this, we explored genes related to genomic integrity and cellular survival, focusing on iPSCs (A53T-PD1) that displayed enhanced colony stability. Our investigation had revealed three candidate genes CCN3, POSTN, and PTHLH that exhibited differential expression levels and potential roles in iPSC stability. Subsequent analyses identified various protein interactions for these candidate genes. POSTN, significantly upregulated in A53T-PD1 iPSC line, showed interactions with extracellular matrix components and potential involvement in Wnt signaling. CCN3, also highly upregulated, demonstrated interactions with TP53, CDKN1A, and factors related to apoptosis and proliferation. PTHLH, while upregulated, exhibited interactions with CDK2 and genes involved in cell cycle regulation. RT-qPCR validation confirmed elevated CCN3 and PTHLH expression in A53T-PD1 iPSCs, aligning with RNA-seq findings. These genes' roles in preserving pluripotency and cellular stability require further exploration. In conclusion, we identified CCN3, POSTN, and PTHLH as potential contributors to genomic integrity and pluripotency maintenance in iPSCs. Their roles in DNA repair, apoptosis evasion, and signaling pathways could offer valuable insights for enhancing reprogramming efficiency and sustaining pluripotency. Further investigations are essential to unravel the mechanisms underlying their actions.
Collapse
Affiliation(s)
- Nuha T. Swaidan
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Nada H. Soliman
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Ahmed T. Aboughalia
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Toqa Darwish
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Ruba O. Almeshal
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Azhar A. Al-Khulaifi
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Rowaida Z. Taha
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Rania Alanany
- College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar
| | | | - Salam Salloum-Asfar
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Sara A. Abdulla
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Abdallah M. Abdallah
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Mohamed M. Emara
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| |
Collapse
|
|
10
|
Aich M, Ansari AH, Ding L, Iesmantavicius V, Paul D, Choudhary C, Maiti S, Buchholz F, Chakraborty D. TOBF1 modulates mouse embryonic stem cell fate through regulating alternative splicing of pluripotency genes. Cell Rep 2023; 42:113177. [PMID: 37751355 DOI: 10.1016/j.celrep.2023.113177] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2023] [Revised: 06/28/2023] [Accepted: 09/11/2023] [Indexed: 09/28/2023] Open
Abstract
Embryonic stem cells (ESCs) can undergo lineage-specific differentiation, giving rise to different cell types that constitute an organism. Although roles of transcription factors and chromatin modifiers in these cells have been described, how the alternative splicing (AS) machinery regulates their expression has not been sufficiently explored. Here, we show that the long non-coding RNA (lncRNA)-associated protein TOBF1 modulates the AS of transcripts necessary for maintaining stem cell identity in mouse ESCs. Among the genes affected is serine/arginine splicing factor 1 (SRSF1), whose AS leads to global changes in splicing and expression of a large number of downstream genes involved in the maintenance of ESC pluripotency. By overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and AS upon its knockout, we describe local nuclear territories where these distinct events converge. Collectively, these contribute to the maintenance of mouse ESC identity.
Collapse
Affiliation(s)
- Meghali Aich
- CSIR- Institute of Genomics and Integrative Biology, New Delhi 110025, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Asgar Hussain Ansari
- CSIR- Institute of Genomics and Integrative Biology, New Delhi 110025, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Li Ding
- Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany
| | - Vytautas Iesmantavicius
- The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Deepanjan Paul
- CSIR- Institute of Genomics and Integrative Biology, New Delhi 110025, India
| | - Chunaram Choudhary
- The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Souvik Maiti
- CSIR- Institute of Genomics and Integrative Biology, New Delhi 110025, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Frank Buchholz
- Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany
| | - Debojyoti Chakraborty
- CSIR- Institute of Genomics and Integrative Biology, New Delhi 110025, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
| |
Collapse
|
|
11
|
Martin-Vega A, Cobb MH. Navigating the ERK1/2 MAPK Cascade. Biomolecules 2023; 13:1555. [PMID: 37892237 PMCID: PMC10605237 DOI: 10.3390/biom13101555] [Citation(s) in RCA: 39] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Revised: 10/16/2023] [Accepted: 10/18/2023] [Indexed: 10/29/2023] Open
Abstract
The RAS-ERK pathway is a fundamental signaling cascade crucial for many biological processes including proliferation, cell cycle control, growth, and survival; common across all cell types. Notably, ERK1/2 are implicated in specific processes in a context-dependent manner as in stem cells and pancreatic β-cells. Alterations in the different components of this cascade result in dysregulation of the effector kinases ERK1/2 which communicate with hundreds of substrates. Aberrant activation of the pathway contributes to a range of disorders, including cancer. This review provides an overview of the structure, activation, regulation, and mutational frequency of the different tiers of the cascade; with a particular focus on ERK1/2. We highlight the importance of scaffold proteins that contribute to kinase localization and coordinate interaction dynamics of the kinases with substrates, activators, and inhibitors. Additionally, we explore innovative therapeutic approaches emphasizing promising avenues in this field.
Collapse
Affiliation(s)
- Ana Martin-Vega
- Department of Pharmacology, UT Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390, USA;
| | - Melanie H. Cobb
- Department of Pharmacology, UT Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390, USA;
- Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390, USA
| |
Collapse
|
|
12
|
Sánchez-Duffhues G, Hiepen C. Human iPSCs as Model Systems for BMP-Related Rare Diseases. Cells 2023; 12:2200. [PMID: 37681932 PMCID: PMC10487005 DOI: 10.3390/cells12172200] [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: 07/18/2023] [Revised: 08/17/2023] [Accepted: 08/23/2023] [Indexed: 09/09/2023] Open
Abstract
Disturbances in bone morphogenetic protein (BMP) signalling contribute to onset and development of a number of rare genetic diseases, including Fibrodysplasia ossificans progressiva (FOP), Pulmonary arterial hypertension (PAH), and Hereditary haemorrhagic telangiectasia (HHT). After decades of animal research to build a solid foundation in understanding the underlying molecular mechanisms, the progressive implementation of iPSC-based patient-derived models will improve drug development by addressing drug efficacy, specificity, and toxicity in a complex humanized environment. We will review the current state of literature on iPSC-derived model systems in this field, with special emphasis on the access to patient source material and the complications that may come with it. Given the essential role of BMPs during embryonic development and stem cell differentiation, gain- or loss-of-function mutations in the BMP signalling pathway may compromise iPSC generation, maintenance, and differentiation procedures. This review highlights the need for careful optimization of the protocols used. Finally, we will discuss recent developments towards complex in vitro culture models aiming to resemble specific tissue microenvironments with multi-faceted cellular inputs, such as cell mechanics and ECM together with organoids, organ-on-chip, and microfluidic technologies.
Collapse
Affiliation(s)
- Gonzalo Sánchez-Duffhues
- Nanomaterials and Nanotechnology Research Center (CINN-CSIC), ISPA-HUCA, Avda. de Roma, s/n, 33011 Oviedo, Spain
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
| | - Christian Hiepen
- Department of Engineering and Natural Sciences, Westphalian University of Applied Sciences, August-Schmidt-Ring 10, 45665 Recklinghausen, Germany
| |
Collapse
|
|
13
|
Sampayo RG, Sakamoto M, Wang M, Kumar S, Schaffer DV. Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases. Proc Natl Acad Sci U S A 2023; 120:e2219854120. [PMID: 37216516 PMCID: PMC10235963 DOI: 10.1073/pnas.2219854120] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Accepted: 04/24/2023] [Indexed: 05/24/2023] Open
Abstract
During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.
Collapse
Affiliation(s)
- Rocío G. Sampayo
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Mason Sakamoto
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Madeline Wang
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Sanjay Kumar
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - David V. Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| |
Collapse
|
|
14
|
Wnt signaling and the regulation of pluripotency. Curr Top Dev Biol 2023; 153:95-119. [PMID: 36967203 DOI: 10.1016/bs.ctdb.2023.01.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
The role of Wnt signaling in stem cells has been mired in seemingly contradictory findings. On one hand, Wnt has been heralded as a self-renewal factor. On the other hand, Wnt's association with differentiation and lineage commitment is indisputable. This apparent contradiction is particularly evident in pluripotent stem cells, where Wnt promotes self-renewal as well as differentiation. To resolve this discrepancy one must delve into fundamental principles of pluripotency and gain an appreciation for the concept of pluripotency states, which exist in a continuum with intermediate metastable states, some of which have been stabilized in vitro. Wnt signaling is a critical regulator of transitions between pluripotent states. Here, we will discuss Wnt's roles in maintaining pluripotency, promoting differentiation, as well as stimulating reprogramming of somatic cells to an induced pluripotent state.
Collapse
|
|
15
|
Pokhrel N, Genin O, Sela-Donenfeld D, Cinnamon Y. Storage temperature dictates the ability of chicken embryos to successfully resume development by regulating expression of blastulation and gastrulation genes. Front Physiol 2022; 13:960061. [PMID: 36589431 PMCID: PMC9800875 DOI: 10.3389/fphys.2022.960061] [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: 06/02/2022] [Accepted: 11/25/2022] [Indexed: 12/23/2022] Open
Abstract
The avian embryo has a remarkable ability that allows it to suspend its development during blastulation for a long time at low temperatures, and to resume normal development when incubated. This ability is used by poultry hatcheries to store eggs prior to incubation. We have previously found that this ability correlates with the temperature during storage; embryos recover much better following prolonged storage at 12°C rather than at 18°C. However, the molecular and cellular mechanisms underlying these differences are poorly understood. To successfully resume development following storage, the embryo has to shift from the blastulation phase to gastrulation. Several genes are known to partake in the blastulation-to-gastrulation transition under normal conditions, such as the pluripotency-related genes Inhibitor of DNA Binding 2 (ID2) and NANOG that are expressed during blastulation, and the gastrulation-regulating genes NODAL and Brachyury (TBXT). However, their expression and activity following storage is unknown. To elucidate the molecular mechanisms that initiate the ability to successfully transit from blastulation to gastrulation following storage, embryos were stored for 28 days at 12°C or 18°C, and were assessed either prior to incubation, 12, or 18 h of incubation at 37.8°C. Immediately following storage at 18°C group showed remarkable impaired morphology compared to the blastoderm of the 12°C group and of non-stored control embryos. Concurrently with these, expression of ID2 and NANOG was maintained following storage at 12°C similar to the control group, but was significantly reduced upon storage at 18°C. Nevertheless, when the 18°C-stored embryos were incubated, the morphology and the reduced genes were reverted to resemble those of the 12°C group. At variance, key gastrulation genes, NODAL and its downstream effector Brachyury (TBXT), which were similarly expressed in the control and the 12°C group, were not restored in the 18°C embryos following incubation. Notably, ectopic administration of Activin rescued NODAL and TBXT expression in the 18°C group, indicating that these embryos maintain the potential to initiate. Collectively, this study suggests a temperature-dependent mechanisms that direct the transition from blastulation to gastrulation. These mechanisms promote a successful developmental resumption following prolonged storage at low temperatures.
Collapse
Affiliation(s)
- Narayan Pokhrel
- Agriculture Research Organization, Volcani Center, Department of Poultry and Aquaculture Science, Rishon LeTsiyon, Israel,Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
| | - Olga Genin
- Agriculture Research Organization, Volcani Center, Department of Poultry and Aquaculture Science, Rishon LeTsiyon, Israel
| | - Dalit Sela-Donenfeld
- Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel,*Correspondence: Dalit Sela-Donenfeld, ; Yuval Cinnamon,
| | - Yuval Cinnamon
- Agriculture Research Organization, Volcani Center, Department of Poultry and Aquaculture Science, Rishon LeTsiyon, Israel,*Correspondence: Dalit Sela-Donenfeld, ; Yuval Cinnamon,
| |
Collapse
|
|
16
|
Bone morphogenetic protein 4 rescues the bone regenerative potential of old muscle-derived stem cells via regulation of cell cycle inhibitors. Stem Cell Res Ther 2022; 13:385. [PMID: 35907860 PMCID: PMC9338549 DOI: 10.1186/s13287-022-03047-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 07/11/2022] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Bone morphogenetic protein 4 (BMP4) promotes the osteogenic differentiation and the bone regenerative potential of muscle-derived stem cells (MDSCs). BMP4 also promotes the self-renewal of both embryonic and somatic stem cells; however, BMP4 signaling activity significantly decreases with age. Cyclin-dependent kinase inhibitors P16INK4A (P16) and P18INK4C (P18) induce early G1-phase cell cycle blockade by targeting cyclin-dependent kinase 4/6. It is still unclear if BMP4 affects the bone regenerative potential of old MDSCs through regulation of P16 and P18 expression. METHODS Young and old MDSCs were isolated from 3 week (young) and 2-year-old (old) mice. In vitro cell proliferation and multipotent differentiation were performed for young and old MDSCs both before and after BMP4/GFP transduction. Cell cycle genes were analyzed using Q-PCR. The bone regenerative potential of young and old MDSCs transduced with BMP4/GFP were compared using Micro-CT and histological analysis. The bone regenerative potential of young and old MDSCs was also compared between single and double transduction (higher BMP4 levels expression). The cell proliferation, mitochondrial function and osteogenic differentiation was also compared in vitro between cells that have been transduced with BMP4GFP (single and double transduction). The correlation of bone regeneration capacity of young and old MDSCs with P16 and P18 expression was further evaluated at 10 days after cell transplantation using histology and western blot analysis. RESULTS Old murine MDSCs (MDSCs) exhibit reduced proliferation and multi-lineage differentiation potential with or without BMP4 stimulation, when compared to young murine MDSCs. Old MDSCs express significantly higher P16 and lower P18, with more cells in the G0/1 phase and fewer cells in the G2/M phase, compared to young MDSCs. Old MDSCs retrovirally transduced to express BMP4 regenerated less bone in a critical size skull defect in CD-1 nude mice when compared to young retrovirally transduced MDSCs expressing similar BMP4 levels and contribute less to the new regenerated new bone. Importantly, both young and old MDSCs can regenerate more bone when BMP4 expression levels are increased by double-transduction with the retroviral-BMP4/GFP. However, the bone regeneration enhancement with elevated BMP4 was more profound in old MDSCs (400% at 2 weeks) compared to young MDSCs (200%). Accordingly, P18 is upregulated while P16 is downregulated after BMP4 transduction. Double transduction did not further increase cell proliferation nor mitochondrial function but did significantly increase Osx expression in both young and old MDSCs. Old MDSCs had even significant higher Osx levels as compared to young MDSCs following double transduction, while a similar Alp expression was observed between young and old MDSCs after double transduction. In addition, at 10 days after cell transplantation, old MDSCs having undergone double transduction regenerated bone more rapidly as showed by Alcian blue and Von Kossa staining. Western blot assays demonstrated that old MDSCs after retro-BMP4/GFP double transduction have significantly lower P18 expression levels when compared to young BMP4-transduced MDSCs. In addition, P18 expression was slightly increased in old MDSCs after double transduction when compared to single transduction. P16 expression was not detectable for both young and two old BMP4/GFP transduced MDSCs groups. CONCLUSIONS In summary, BMP4 can offset the adverse effect of aging on the osteogenic differentiation and the bone regenerative potential of old MDSCs via up-regulation of P18 and down-regulation P16 expression.
Collapse
|
|
17
|
Chen G, Yin S, Zeng H, Li H, Wan X. Regulation of Embryonic Stem Cell Self-Renewal. LIFE (BASEL, SWITZERLAND) 2022; 12:life12081151. [PMID: 36013330 PMCID: PMC9410528 DOI: 10.3390/life12081151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 07/12/2022] [Accepted: 07/21/2022] [Indexed: 11/16/2022]
Abstract
Embryonic stem cells (ESCs) are a type of cells capable of self-renewal and multi-directional differentiation. The self-renewal of ESCs is regulated by factors including signaling pathway proteins, transcription factors, epigenetic regulators, cytokines, and small molecular compounds. Similarly, non-coding RNAs, small RNAs, and microRNAs (miRNAs) also play an important role in the process. Functionally, the core transcription factors interact with helper transcription factors to activate the expression of genes that contribute to maintaining pluripotency, while suppressing the expression of differentiation-related genes. Additionally, cytokines such as leukemia suppressor factor (LIF) stimulate downstream signaling pathways and promote self-renewal of ESCs. Particularly, LIF binds to its receptor (LIFR/gp130) to trigger the downstream Jak-Stat3 signaling pathway. BMP4 activates the downstream pathway and acts in combination with Jak-Stat3 to promote pluripotency of ESCs in the absence of serum. In addition, activation of the Wnt-FDZ signaling pathway has been observed to facilitate the self-renewal of ESCs. Small molecule modulator proteins of the pathway mentioned above are widely used in in vitro culture of stem cells. Multiple epigenetic regulators are involved in the maintenance of ESCs self-renewal, making the epigenetic status of ESCs a crucial factor in this process. Similarly, non-coding RNAs and cellular energetics have been described to promote the maintenance of the ESC's self-renewal. These factors regulate the self-renewal and differentiation of ESCs by forming signaling networks. This review focused on the role of major transcription factors, signaling pathways, small molecular compounds, epigenetic regulators, non-coding RNAs, and cellular energetics in ESC's self-renewal.
Collapse
Affiliation(s)
- Guofang Chen
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
| | - Shasha Yin
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
| | - Hongliang Zeng
- Institute of Chinese Materia Medica, Hunan Academy of Chinese Medicine, Changsha 410013, China;
| | - Haisen Li
- School of Medicine, Wayne State University, Detroit, MI 48201, USA
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
| | - Xiaoping Wan
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
| |
Collapse
|
|
18
|
Roberts RM, Ezashi T, Temple J, Owen JR, Soncin F, Parast MM. The role of BMP4 signaling in trophoblast emergence from pluripotency. Cell Mol Life Sci 2022; 79:447. [PMID: 35877048 PMCID: PMC10243463 DOI: 10.1007/s00018-022-04478-w] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Revised: 05/24/2022] [Accepted: 07/06/2022] [Indexed: 11/03/2022]
Abstract
The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.
Collapse
Affiliation(s)
- R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
| | - Toshihiko Ezashi
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
- Colorado Center for Reproductive Medicine, 10290 Ridgegate Circle, Lone Tree, CO, 80124, USA
| | - Jasmine Temple
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
- Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA
| | - Joseph R Owen
- Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Diego, La Jolla, CA, USA
| | - Francesca Soncin
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
- Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA
| | - Mana M Parast
- Department of Pathology, University of California San Diego, La Jolla, CA, USA.
- Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA.
| |
Collapse
|
|
19
|
Wesseler F, Riege D, Puthanveedu M, Halver J, Müller E, Bertrand J, Antonchick AP, Sievers S, Waldmann H, Schade D. Probing Embryonic Development Enables the Discovery of Unique Small-Molecule Bone Morphogenetic Protein Potentiators. J Med Chem 2022; 65:3978-3990. [PMID: 35108017 DOI: 10.1021/acs.jmedchem.1c01800] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We report on the feasibility to harness embryonic development in vitro for the identification of small-molecule cytokine mimetics and signaling activators. Here, a phenotypic, target-agnostic, high-throughput assay is presented that probes bone morphogenetic protein (BMP) signaling during mesodermal patterning of embryonic stem cells. The temporal discrimination of BMP- and transforming growth factor-β (TGFβ)-driven stages of cardiomyogenesis underpins a selective, authentic orchestration of BMP cues that can be recapitulated for the discovery of BMP activator chemotypes. Proof of concept is shown from a chemical screen of 7000 compounds, provides a robust hit validation workflow, and afforded 2,3-disubstituted 4H-chromen-4-ones as potent BMP potentiators with osteogenic efficacy. Mechanistic studies suggest that Chromenone 1 enhances canonical BMP outputs at the expense of TGFβ-Smads in an unprecedented manner. Pharmacophoric features were defined, providing a set of novel chemical probes for various applications in (stem) cell biology, regenerative medicine, and basic research on the BMP pathway.
Collapse
Affiliation(s)
- Fabian Wesseler
- Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany.,Compound Management and Screening Center, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.,Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany
| | - Daniel Riege
- Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany
| | - Mahesh Puthanveedu
- Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany.,Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
| | - Jonas Halver
- Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
| | - Eva Müller
- Department of Orthopedic Surgery, Otto-von-Guericke University, 39120 Magdeburg, Germany
| | - Jessica Bertrand
- Department of Orthopedic Surgery, Otto-von-Guericke University, 39120 Magdeburg, Germany
| | - Andrey P Antonchick
- Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany.,Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.,Department of Chemistry and Forensics, College of Science and Technology, Nottingham Trent University, Clifton Lane, NG11 8NS Nottingham, United Kingdom
| | - Sonja Sievers
- Compound Management and Screening Center, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.,Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
| | - Herbert Waldmann
- Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany.,Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
| | - Dennis Schade
- Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany.,Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.,Partner Site Kiel, DZHK, German Center for Cardiovascular Research, 24105 Kiel, Germany
| |
Collapse
|
|
20
|
Fukihara J, Maiolo S, Kovac J, Sakamoto K, Wakahara K, Hashimoto N, Reynolds PN. Overexpression of bone morphogenetic protein receptor type 2 suppresses transforming growth factor β-induced profibrotic responses in lung fibroblasts. Exp Lung Res 2022; 48:35-51. [PMID: 35037801 DOI: 10.1080/01902148.2021.2024301] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
MATERIALS AND METHODS We investigated BMPR2 expression in pulmonary fibrosis and TGF-β/BMP signaling in lung fibroblasts. Then we evaluated the impact of BMPR2 upregulation using adenoviral transduction on TGF-β-induced Smad2/3 phosphorylation and fibronectin production in lung fibroblasts. RESULTS BMPR2 was distributed in airway epithelium and alveolar walls in rat lungs. BMPR2 expression was decreased in fibrotic lesions in the lungs of rats with bleomycin-induced pulmonary fibrosis and in human lung fibroblasts (HLFs) stimulated with TGF-β. Although Smad2/3 phosphorylation and fibronectin production were not suppressed solely by BMPs, phosphorylated Smad2/3 was decreased in BMPR2-transduced cells even without BMP stimulation. Fibronectin was decreased only when BMPR2-transduced HLFs were stimulated with BMP7 (but not BMP4). Similar results were also observed in IPF patient HLFs and rat lung fibroblasts. CONCLUSIONS BMPR2 expression was reduced in fibrotic lungs and lung fibroblasts stimulated with TGF-β. BMPR2 transduction to lung fibroblasts reduced Smad2/3 phosphorylation, and reduced fibronectin production when treated with BMP7. Upregulation of BMPR2 may be a possible strategy for treating pulmonary fibrosis.
Collapse
Affiliation(s)
- Jun Fukihara
- Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.,Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia
| | - Suzanne Maiolo
- Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia
| | - Jessica Kovac
- Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia
| | - Koji Sakamoto
- Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Keiko Wakahara
- Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Naozumi Hashimoto
- Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Paul N Reynolds
- Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia.,Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia
| |
Collapse
|
|
21
|
Colonnetta MM, Goyal Y, Johnson HE, Syal S, Schedl P, Deshpande G. Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo. PLoS Genet 2022; 18:e1010002. [PMID: 34986144 PMCID: PMC8765614 DOI: 10.1371/journal.pgen.1010002] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 01/18/2022] [Accepted: 12/17/2021] [Indexed: 11/24/2022] Open
Abstract
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate. Proper specification of primordial germ cells (PGCs) is crucial as PGCs serve as the precursors of germline stem cells. To specify PGC fate, invertebrates rely upon cell autonomous preformation involving maternally deposited germ plasm. In Drosophila melanogaster, to insulate newly formed PGCs from the adverse effects of the cell-cell signaling pathways, germ plasm determinants silence transcription and attenuate the cell cycle. However, our data on the BMP signaling pathway challenge this long-held view of PGC specification and suggest that appropriate specification of embryonic PGCs is sensitive to the BMP ligand, decapentaplegic (dpp), and its cognate receptor, thickveins. We find that PGCs are not only capable of responding to BMP signals from the soma, but also that these signals impact the proper determination of the germ cells. Based on these unanticipated similarities between mammals and flies, we propose a model integrating contribution of both the cell-autonomous (preformation) and non-autonomous (epigenesis) pathways during PGC determination. Consistent with the model, we have observed dominant genetic interactions between, oskar, the maternal determinant of PGC fate, and the BMP pathway ligand dpp.
Collapse
Affiliation(s)
- Megan M. Colonnetta
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Yogesh Goyal
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Heath E. Johnson
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Sapna Syal
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Paul Schedl
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Girish Deshpande
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
- * E-mail:
| |
Collapse
|
|
22
|
Pluripotency Stemness and Cancer: More Questions than Answers. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1376:77-100. [PMID: 34725790 DOI: 10.1007/5584_2021_663] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.
Collapse
|
|
23
|
Iozon S, Caracostea GV, Páll E, Şoriţău O, Mănăloiu ID, Bulboacă AE, Lupşe M, Mihu CM, Roman AL. Injectable platelet-rich fibrin influences the behavior of gingival mesenchymal stem cells. ROMANIAN JOURNAL OF MORPHOLOGY AND EMBRYOLOGY 2021; 61:189-198. [PMID: 32747910 PMCID: PMC7728122 DOI: 10.47162/rjme.61.1.21] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
In this study, we examined the effects of injectable platelet-rich fibrin (iPRF) on proliferation and osteodifferentiation in mesenchymal stem cells (MSCs) isolated from human gingiva. Gingival MSCs (gMSCs) were grown in experimental culture media with different concentrations of iPRF [5%, 10%, and replacement of fetal calf serum (FCS) in the standard media with 10% iPRF–10% iPRF-FCS]. Immunophenotyping of gMSCs was performed after seven days by flow cytometry, and their proliferation was examined after three and seven days using the Cell Counting Kit-8 method. After 14 days in culture, spontaneous osteogenic differentiation of gMSCs was evaluated via real-time polymerase chain reaction. All gMSCs were positive for cluster of differentiation (CD) 105, CD73, CD90, and CD44, and negative for CD34/45, CD14, CD79a, and human leukocyte antigen, DR isotype (HLA-DR). Reduced expression of some surface antigens was observed in the gMSCs grown in 10% iPRF-FCS medium compared to the other groups. After three days, gMSCs grown in 10% iPRF had proliferated significantly less than the other groups. After seven days, proliferation was significantly higher in the 5% iPRF cells compared to the control, while proliferation in the 10% iPRF and 10% iPRF-FCS groups was significantly lower. No spontaneous osteogenic differentiation was observed in the presence of iPRF, as observed by low runt-related transcription factor 2 (RUNX2) expression. Some expression of secreted protein acidic and cysteine rich (SPARC) and collagen 1 alpha (COL1A) was observed for all the gMSCs regardless of the culture medium composition. gMSCs grown in 10% iPRF had significantly lower SPARC expression. In conclusion, 5% iPRF stimulated gMSC proliferation, and an excessively high concentration of iPRF can impair osteogenic induction.
Collapse
Affiliation(s)
- Sofia Iozon
- Discipline of Histology, Department of Morphological Sciences, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania;
| | | | | | | | | | | | | | | | | |
Collapse
|
|
24
|
Raghav PK, Kalyanaraman K, Kumar D. Human cell receptors: potential drug targets to combat COVID-19. Amino Acids 2021; 53:813-842. [PMID: 33950300 PMCID: PMC8097256 DOI: 10.1007/s00726-021-02991-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Accepted: 04/21/2021] [Indexed: 01/08/2023]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) has announced that COVID-19 is a pandemic having a higher spread rate rather than the mortality. Identification of a potential approach or therapy against COVID-19 is still under consideration. Therefore, it is essential to have an insight into SARS-CoV-2, its interacting partner, and domains for an effective treatment. The present study is divided into three main categories, including SARS-CoV-2 prominent receptor and its expression levels, other interacting partners, and their binding domains. The first section focuses primarily on coronaviruses' general aspects (SARS-CoV-2, SARS-CoV, and the Middle East Respiratory Syndrome Coronaviruses (MERS-CoV)) their structures, similarities, and mode of infections. The second section discusses the host receptors which includes the human targets of coronaviruses like dipeptidyl peptidase 4 (DPP4), CD147, CD209L, Angiotensin-Converting Enzyme 2 (ACE2), and other miscellaneous targets (type-II transmembrane serine proteases (TTSPs), furin, trypsin, cathepsins, thermolysin, elastase, phosphatidylinositol 3-phosphate 5-kinase, two-pore segment channel, and epithelium sodium channel C-α subunit). The human cell receptor, ACE2 plays an essential role in the Renin-Angiotensin system (RAS) pathway and COVID-19. Thus, this section also discusses the ACE2 expression and risk of COVID-19 infectivity in various organs and tissues such as the liver, lungs, intestine, heart, and reproductive system in the human body. Absence of ACE2 protein expression in immune cells could be used for limiting the SARS-CoV-2 infection. The third section covers the current available approaches for COVID-19 treatment. Overall, this review focuses on the critical role of human cell receptors involved in coronavirus pathogenesis, which would likely be used in designing target-specific drugs to combat COVID-19.
Collapse
Affiliation(s)
| | - Keerthana Kalyanaraman
- Amity Institute of Biotechnology, Amity University, Sector-125, Noida, Uttar Pradesh, India
| | - Dinesh Kumar
- ICMR-National Institute of Cancer Prevention & Research, Noida, 201301, India.
| |
Collapse
|
|
25
|
Zhao X, Bian R, Wang F, Wang Y, Li X, Guo Y, Zhang X, Luo G, Zhan R. GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling. Stem Cell Res Ther 2021; 12:42. [PMID: 33413682 PMCID: PMC7792190 DOI: 10.1186/s13287-020-02106-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 12/15/2020] [Indexed: 01/08/2023] Open
Abstract
OBJECTIVE Epidermal stem cells (EpSCs) can self-renew, which are responsible for the long-term maintenance of the skin, and it also plays a critical role in wound re-epithelization, but the mechanism underlying EpSCs proliferation is unclear. GDF-5, also known as BMP-14, is a member of the BMP family and can be used as a self-renewal supporter. Here, we studied the effects of GDF-5 on mouse EpSCs proliferation mechanism in wound healing. METHODS Firstly, the effects of GDF-5 on EpSCs proliferation was tested by using CCK8 reagent and PCNA expression was analyzed by Western blotting. Secondly, we screened genes that promote EpSCs proliferation in the FOX and cyclin family by qPCR, and then the protein expression level of the selected genes was further analyzed by Western blotting. Thirdly, siRNA plasmids and pAdEasy adenovirus were transfected or infected, respectively, into mouse EpSCs to detect the effect of target genes on GDF-5-induced cell proliferation. Furthermore, we injected GDF-5 to a deep partial thickness burn mouse model for finding out whether EpSCs proliferation can be detected by immunohistochemical. Finally, the relevant target genes were analyzed by qPCR, immunoblotting, and dual-luciferase reporter gene detection. RESULTS We discovered that 100 ng/ml recombinant mouse GDF-5 was the optimal concentration for promoting mouse EpSCs proliferation. Through preliminary screened by qPCR, we found that Foxg1 and cyclin D1 could be the downstream molecules of GDF-5, and the results were confirmed by Western blotting. And the effect of GDF-5 on mouse EpSCs proliferation was adjusted by Foxg1/cyclin D1 in vitro and in vivo. Besides, GDF-5-induced transcription of cyclin D1 was regulated by Foxg1-mediated cyclin D1 promoter activity. CONCLUSION This paper showed that GDF-5 promotes mouse EpSCs proliferation via Foxg1-cyclin D1 signal pathway. It is suggested that GDF-5 may be a new approach to make EpSCs proliferation which can be used in wound healing.
Collapse
Affiliation(s)
- Xiaohong Zhao
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Ruyu Bian
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Fan Wang
- Department of Plastic and Reconstructive Surgery, Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Ying Wang
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Xue Li
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Yicheng Guo
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Xiaorong Zhang
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Gaoxing Luo
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China.
| | - Rixing Zhan
- Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China.
| |
Collapse
|
|
26
|
Perera SN, Kerosuo L. On the road again: Establishment and maintenance of stemness in the neural crest from embryo to adulthood. STEM CELLS (DAYTON, OHIO) 2020; 39:7-25. [PMID: 33017496 PMCID: PMC7821161 DOI: 10.1002/stem.3283] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 09/08/2020] [Accepted: 09/11/2020] [Indexed: 12/22/2022]
Abstract
Unique to vertebrates, the neural crest (NC) is an embryonic stem cell population that contributes to a greatly expanding list of derivatives ranging from neurons and glia of the peripheral nervous system, facial cartilage and bone, pigment cells of the skin to secretory cells of the endocrine system. Here, we focus on what is specifically known about establishment and maintenance of NC stemness and ultimate fate commitment mechanisms, which could help explain its exceptionally high stem cell potential that exceeds the "rules set during gastrulation." In fact, recent discoveries have shed light on the existence of NC cells that coexpress commonly accepted pluripotency factors like Nanog, Oct4/PouV, and Klf4. The coexpression of pluripotency factors together with the exceptional array of diverse NC derivatives encouraged us to propose a new term "pleistopotent" (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post-gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC-derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos.
Collapse
Affiliation(s)
- Surangi N Perera
- Neural Crest Development and Disease Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
| | - Laura Kerosuo
- Neural Crest Development and Disease Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
| |
Collapse
|
|
27
|
Yang YY, Lin CJ, Wang CC, Chen CM, Kao WJ, Chen YH. Consecutive Hypoxia Decreases Expression of NOTCH3, HEY1, CC10, and FOXJ1 via NKX2-1 Downregulation and Intermittent Hypoxia-Reoxygenation Increases Expression of BMP4, NOTCH1, MKI67, OCT4, and MUC5AC via HIF1A Upregulation in Human Bronchial Epithelial Cells. Front Cell Dev Biol 2020; 8:572276. [PMID: 33015064 PMCID: PMC7500169 DOI: 10.3389/fcell.2020.572276] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Accepted: 08/17/2020] [Indexed: 01/11/2023] Open
Abstract
Previous studies have shown that the experimental models of hypoxia-reoxygenation (H/R) mimics the physiological conditions of ischemia-reperfusion and induce oxidative stress and injury in various types of organs, tissues, and cells, both in vivo and in vitro, including human lung adenocarcinoma epithelial cells. Nonetheless, it had not been reported whether H/R affected proliferation, apoptosis, and expression of stem/progenitor cell markers in the bronchial epithelial cells. In this study, we investigated differential effects of consecutive hypoxia and intermittent 24/24-h cycles of H/R on human bronchial epithelial (HBE) cells derived from the same-race and age-matched healthy subjects (i.e., NHBE) and subjects with chronic obstructive pulmonary disease (COPD) (i.e., DHBE). To analyze gene/protein expression during differentiation, both the NHBE and DHBE cells at the 2nd passage were cultured at the air-liquid interface (ALI) in the differentiation medium under normoxia for 3 days, followed by either culturing under hypoxia (1% O2) for consecutively 9 days and then returning to normoxia for another 9 days, or culturing under 24/24-h cycles of H/R (i.e., 24 h of 1% O2 followed by 24 h of 21% O2, repetitively) for 18 days in total, so that all differentiating HBE cells were exposed to hypoxia for a total of 9 days. In both the normal and diseased HBE cells, intermittent H/R significantly increased HIF1A, BMP4, NOTCH1, MKI67, OCT4, and MUC5AC expression, while consecutive hypoxia significantly decreased NKX2-1, NOTCH3, HEY1, CC10, and FOXJ1 expression. Inhibition of HIF1A or NKX2-1 expression by siRNA transfection respectively decreased BMP4/NOTCH1/MKI67/OCT4/MUC5AC and NOTCH3/HEY1/CC10/FOXJ1 expression in the HBE cells cultured under intermittent H/R to the same levels under normoxia. Overexpression of NKX2-1 via cDNA transfection caused more than 2.8-fold increases in NOTCH3, HEY1, and FOXJ1 mRNA levels in the HBE cells cultured under consecutive hypoxia compared to the levels under normoxia. Taken together, our results show for the first time that consecutive hypoxia decreased expression of the co-regulated gene module NOTCH3/HEY1/CC10 and the ciliogenesis-inducing transcription factor gene FOXJ1 via NKX2-1 mRNA downregulation, while intermittent H/R increased expression of the co-regulated gene module BMP4/NOTCH1/MKI67/OCT4 and the predominant airway mucin gene MUC5AC via HIF1A mRNA upregulation.
Collapse
Affiliation(s)
- Yung-Yu Yang
- Department of General Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Chao-Ju Lin
- Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan
| | - Cheng-Chin Wang
- Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan.,Section of Respiratory Therapy, Rueifang Miner Hospital, New Taipei City, Taiwan
| | - Chieh-Min Chen
- Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
| | - Wen-Jen Kao
- Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan
| | - Yi-Hui Chen
- Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan
| |
Collapse
|
|
28
|
CRH Promotes the Neurogenic Activity of Neural Stem Cells in the Adult Hippocampus. Cell Rep 2020; 29:932-945.e7. [PMID: 31644914 DOI: 10.1016/j.celrep.2019.09.037] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2017] [Revised: 01/17/2018] [Accepted: 09/12/2019] [Indexed: 02/07/2023] Open
Abstract
Local cues in the adult neurogenic niches dynamically regulate homeostasis in neural stem cells, whereas their identity and associated molecular mechanisms remain poorly understood. Here, we show that corticotropin-releasing hormone (CRH), the major mediator of mammalian stress response and a key neuromodulator in the adult brain, is necessary for hippocampal neural stem cell (hiNSC) activity under physiological conditions. In particular, we demonstrate functionality of the CRH/CRH receptor (CRHR) system in mouse hiNSCs and conserved expression in humans. Most important, we show that genetic deficiency of CRH impairs hippocampal neurogenesis, affects spatial memory, and compromises hiNSCs' responsiveness to environmental stimuli. These deficits have been partially restored by virus-mediated CRH expression. Additionally, we provide evidence that local disruption of the CRH/CRHR system reduces neurogenesis, while exposure of adult hiNSCs to CRH promotes neurogenic activity via BMP4 suppression. Our findings suggest a critical role of CRH in adult neurogenesis, independently of its stress-related systemic function.
Collapse
|
|
29
|
Abstract
Derivation of induced Pluripotent Stem Cells (iPSCs) by reprogramming somatic cells to a pluripotent state has revolutionized stem cell research. Ensuing this, various groups have used genetic and non-genetic approaches to generate iPSCs from numerous cell types. However, achieving a pluripotent state in most of the reprogramming studies is marred by serious limitations such as low reprogramming efficiency and slow kinetics. These limitations are mainly due to the presence of potent barriers that exist during reprogramming when a mature cell is coaxed to achieve a pluripotent state. Several studies have revealed that intrinsic factors such as non-optimal stoichiometry of reprogramming factors, specific signaling pathways, cellular senescence, pluripotency-inhibiting transcription factors and microRNAs act as a roadblock. In addition, the epigenetic state of somatic cells and specific epigenetic modifications that occur during reprogramming also remarkably impede the generation of iPSCs. In this review, we present a comprehensive overview of the barriers that inhibit reprogramming and the understanding of which will pave the way to develop safe strategies for efficient reprogramming.
Collapse
|
|
30
|
Hahner F, Moll F, Schröder K. NADPH oxidases in the differentiation of endothelial cells. Cardiovasc Res 2020; 116:262-268. [PMID: 31393561 DOI: 10.1093/cvr/cvz213] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Revised: 05/10/2019] [Accepted: 08/08/2019] [Indexed: 12/14/2022] Open
Abstract
The differentiation of stem cells into endothelial cells involves the modulation of highly interconnected metabolic and epigenetic processes. Therefore, the differentiation of endothelial cells is a tightly controlled process, which is adjusted at multiple levels, meaning that even the smallest variation can result in major consequences. Reactive oxygen species (ROS) represent a group of second messengers that can interfere with both metabolic and epigenetic processes. Besides their generation by mitochondria, ROS are produced in a controlled manner by the family of NADPH oxidases. The different members of the NADPH oxidase family produce superoxide anions or hydrogen peroxide. Due to the specific sub-cellular localization of the different NADPH oxidases, ROS are produced at diverse sites in the cell, such as the plasma membrane or the endoplasmic reticulum. Once produced, ROS interfere with proteins, lipids, and DNA to modulate intracellular signal cascades. Accordingly, ROS represent a group of readily available and specifically localized modulators of the highly sophisticated signalling network that eventually leads to the differentiation of stem cells into endothelial cells. This review focuses on the role of NADPH oxidases in the differentiation of stem cells into endothelial cells.
Collapse
Affiliation(s)
- Fabian Hahner
- Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, 60590 Frankfurt, Germany
| | - Franziska Moll
- Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, 60590 Frankfurt, Germany
| | - Katrin Schröder
- Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, 60590 Frankfurt, Germany
| |
Collapse
|
|
31
|
Shukla R, Mjoseng HK, Thomson JP, Kling S, Sproul D, Dunican DS, Ramsahoye B, Wongtawan T, Treindl F, Templin MF, Adams IR, Pennings S, Meehan RR. Activation of transcription factor circuity in 2i-induced ground state pluripotency is independent of repressive global epigenetic landscapes. Nucleic Acids Res 2020; 48:7748-7766. [PMID: 32585002 PMCID: PMC7641322 DOI: 10.1093/nar/gkaa529] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Revised: 06/02/2020] [Accepted: 06/15/2020] [Indexed: 01/07/2023] Open
Abstract
Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.
Collapse
Affiliation(s)
- Ruchi Shukla
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
- Newcastle University Centre for Cancer, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne NE2 4HH, UK
| | - Heidi K Mjoseng
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - John P Thomson
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Simon Kling
- NMI Natural and Medical Sciences Institute, Tübingen University, Reutlingen, Germany
| | - Duncan Sproul
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Donncha S Dunican
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Bernard Ramsahoye
- Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Tuempong Wongtawan
- Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Fridolin Treindl
- NMI Natural and Medical Sciences Institute, Tübingen University, Reutlingen, Germany
- Pharmaceutical Biotechnology, Tübingen University, Tübingen, Germany
| | - Markus F Templin
- NMI Natural and Medical Sciences Institute, Tübingen University, Reutlingen, Germany
- Pharmaceutical Biotechnology, Tübingen University, Tübingen, Germany
| | - Ian R Adams
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Sari Pennings
- Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Richard R Meehan
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK
| |
Collapse
|
|
32
|
Taei A, Samadian A, Ghezel-Ayagh Z, Mollamohammadi S, Moradi S, Kiani T, Janzamin E, Farzaneh Z, Tahamtani Y, Braun T, Hassani SN, Baharvand H. Suppression of p38-MAPK endows endoderm propensity to human embryonic stem cells. Biochem Biophys Res Commun 2020; 527:811-817. [PMID: 32446562 DOI: 10.1016/j.bbrc.2020.04.119] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2020] [Accepted: 04/22/2020] [Indexed: 12/28/2022]
Abstract
The ability of human embryonic stem cells (hESCs) to proliferate unlimitedly and give rise to all tissues makes these cells a promising source for cell replacement therapies. To realize the full potential of hESCs in cell therapy, it is necessary to interrogate regulatory pathways that influence hESC maintenance and commitment. Here, we reveal that pharmacological attenuation of p38 mitogen-activated protein kinase (p38-MAPK) in hESCs concomitantly augments some characteristics associated with pluripotency and the expressions of early lineage markers. Moreover, this blockage capacitates hESCs to differentiate towards an endoderm lineage at the expense of other lineages upon spontaneous hESC differentiation. Notably, hESCs pre-treated with p38-MAPK inhibitor exhibit significantly improved pancreatic progenitor directed differentiation. Together, our findings suggest a new approach to the robust endoderm differentiation of hESCs and potentially enables the facile derivation of various endoderm-derived lineages such as pancreatic cells.
Collapse
Affiliation(s)
- Adeleh Taei
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran
| | - Azam Samadian
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Zahra Ghezel-Ayagh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sepideh Mollamohammadi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sharif Moradi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Cancer Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Isar 11, 47138-18983, Babol, Iran
| | - Tahereh Kiani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ehsan Janzamin
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Zahra Farzaneh
- 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 Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran; Department of Diabetes, Obesity, and Metabolism, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Thomas Braun
- Department of Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Seyedeh-Nafiseh Hassani
- Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
| |
Collapse
|
|
33
|
ERK signalling: a master regulator of cell behaviour, life and fate. Nat Rev Mol Cell Biol 2020; 21:607-632. [PMID: 32576977 DOI: 10.1038/s41580-020-0255-7] [Citation(s) in RCA: 643] [Impact Index Per Article: 128.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/07/2020] [Indexed: 12/13/2022]
Abstract
The proteins extracellular signal-regulated kinase 1 (ERK1) and ERK2 are the downstream components of a phosphorelay pathway that conveys growth and mitogenic signals largely channelled by the small RAS GTPases. By phosphorylating widely diverse substrates, ERK proteins govern a variety of evolutionarily conserved cellular processes in metazoans, the dysregulation of which contributes to the cause of distinct human diseases. The mechanisms underlying the regulation of ERK1 and ERK2, their mode of action and their impact on the development and homeostasis of various organisms have been the focus of much attention for nearly three decades. In this Review, we discuss the current understanding of this important class of kinases. We begin with a brief overview of the structure, regulation, substrate recognition and subcellular localization of ERK1 and ERK2. We then systematically discuss how ERK signalling regulates six fundamental cellular processes in response to extracellular cues. These processes are cell proliferation, cell survival, cell growth, cell metabolism, cell migration and cell differentiation.
Collapse
|
|
34
|
Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions. Cells 2020; 9:cells9061349. [PMID: 32485910 PMCID: PMC7349583 DOI: 10.3390/cells9061349] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 05/26/2020] [Accepted: 05/26/2020] [Indexed: 12/19/2022] Open
Abstract
Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.
Collapse
|
|
35
|
The effect of dual inhibition of Ras-MEK-ERK and GSK3β pathways on development of in vitro cultured rabbit embryos. ZYGOTE 2020; 28:183-190. [PMID: 32192548 DOI: 10.1017/s0967199419000753] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Dual inhibition (2i) of Ras-MEK-ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.
Collapse
|
|
36
|
Markovics A, Angyal Á, Tóth KF, Ádám D, Pénzes Z, Magi J, Pór Á, Kovács I, Törőcsik D, Zouboulis CC, Bíró T, Oláh A. GPR119 Is a Potent Regulator of Human Sebocyte Biology. J Invest Dermatol 2020; 140:1909-1918.e8. [PMID: 32142797 DOI: 10.1016/j.jid.2020.02.011] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2018] [Revised: 01/31/2020] [Accepted: 02/12/2020] [Indexed: 12/16/2022]
Abstract
We have shown previously that endocannabinoids promote sebaceous lipogenesis, and sebocytes are involved in the metabolism of the endocannabinoid-like substance oleoylethanolamide (OEA). OEA is an endogenous activator of GPR119, a recently deorphanized receptor, which currently is being investigated as a promising antidiabetic drug target. In this study, we investigated the effects of OEA as well as the expression and role of GPR119 in human sebocytes. We found that OEA promoted differentiation of human SZ95 sebocytes (elevated lipogenesis, enhanced granulation, and the induction of early apoptotic events), and it switched the cells to a proinflammatory phenotype (increased expression and release of several proinflammatory cytokines). Moreover, we could also demonstrate that GPR119 was expressed in human sebocytes, and its small interfering RNA-mediated gene silencing suppressed OEA-induced sebaceous lipogenesis, which was mediated via c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2, protein kinase B, and CRE-binding protein activation. Finally, our pilot data demonstrated that GPR119 was downregulated in the sebaceous glands of patients with acne, arguing that GPR119 signaling may indeed be disturbed in acne. Collectively, our findings introduce the OEA/GPR119 signaling as a positive regulator of sebocyte differentiation and highlight the possibility that dysregulation of this pathway may contribute to the development of seborrhea and acne.
Collapse
Affiliation(s)
- Arnold Markovics
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; University of Debrecen, Doctoral School of Molecular Medicine, Debrecen, Hungary
| | - Ágnes Angyal
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; University of Debrecen, Doctoral School of Molecular Medicine, Debrecen, Hungary
| | - Kinga Fanni Tóth
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; University of Debrecen, Doctoral School of Molecular Medicine, Debrecen, Hungary
| | - Dorottya Ádám
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; University of Debrecen, Doctoral School of Molecular Medicine, Debrecen, Hungary
| | - Zsófia Pénzes
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; University of Debrecen, Doctoral School of Molecular Medicine, Debrecen, Hungary; Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - József Magi
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Ágnes Pór
- Department of Pathology, Gyula Kenézy University Hospital, University of Debrecen, Debrecen, Hungary
| | - Ilona Kovács
- Department of Pathology, Gyula Kenézy University Hospital, University of Debrecen, Debrecen, Hungary
| | - Dániel Törőcsik
- Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Christos C Zouboulis
- Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Brandenburg Medical School Theodor Fontane, Dessau, Germany
| | - Tamás Bíró
- DE-MTA Lendület Cellular Physiology Research Group, Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Attila Oláh
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
| |
Collapse
|
|
37
|
Park S, Han JE, Kim HG, Kim HY, Kim MG, Park JK, Cho GJ, Huang H, Kim MO, Ryoo ZY, Han SH, Choi SK. Inhibition of MAGEA2 regulates pluripotency, proliferation, apoptosis, and differentiation in mouse embryonic stem cells. J Cell Biochem 2020; 121:4667-4679. [PMID: 32065444 DOI: 10.1002/jcb.29692] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Accepted: 01/30/2020] [Indexed: 01/27/2023]
Abstract
Mouse embryonic stem cells (mESCs) exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.
Collapse
Affiliation(s)
- Song Park
- Core Protein Resources Center, DGIST, Daegu, South Korea
| | - Jee Eun Han
- College of Veterinary Medicine, Kyungpook National University, Daegu, South Korea
| | - Hyeon-Gyeom Kim
- School of Life Science, BK21 Plus KNU Creative Bioresearch Group, Kyungpook National University, Daegu, South Korea
| | - Hee-Yeon Kim
- Core Protein Resources Center, DGIST, Daegu, South Korea
| | - Min-Gi Kim
- Core Protein Resources Center, DGIST, Daegu, South Korea
| | - Jin-Kyu Park
- College of Veterinary Medicine, Kyungpook National University, Daegu, South Korea
| | - Gil-Jae Cho
- College of Veterinary Medicine, Kyungpook National University, Daegu, South Korea
| | - Hai Huang
- The School of Animal BT Science, Kyungpook National University, Sangju-si, Gyeongsangbuk-do, South Korea
| | - Myoung Ok Kim
- The School of Animal BT Science, Kyungpook National University, Sangju-si, Gyeongsangbuk-do, South Korea
| | - Zae Young Ryoo
- School of Life Science, BK21 Plus KNU Creative Bioresearch Group, Kyungpook National University, Daegu, South Korea
| | - Se-Hyeon Han
- School of Media Communication, Hanyang University, Seongdonggu, Seoul, South Korea
| | - Seong-Kyoon Choi
- Core Protein Resources Center, DGIST, Daegu, South Korea.,Division of Biotechnology, DGIST, Daegu, South Korea
| |
Collapse
|
|
38
|
Fathi Maroufi N, Hasegawa K, Vahedian V, Nazari Soltan Ahmad S, Zarebkohan A, Miresmaeili Mazrakhondi SA, Hosseini V, Rahbarghazi R. A glimpse into molecular mechanisms of embryonic stem cells pluripotency: Current status and future perspective. J Cell Physiol 2020; 235:6377-6392. [DOI: 10.1002/jcp.29616] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Accepted: 01/09/2020] [Indexed: 12/11/2022]
Affiliation(s)
- Nazila Fathi Maroufi
- Stem Cell and Regenerative Medicine InstituteTabriz University of Medical Sciences Tabriz Iran
- Student Research CommitteeTabriz University of Medical Sciences Tabriz Iran
- Department of Biochemistry and Clinical Laboratories, Faculty of MedicineTabriz University of Medical Sciences Tabriz Iran
| | - Kouichi Hasegawa
- Institute for Integrated Cell‐Material Sciences, Institute for Advanced StudyKyoto University Kyoto Japan
| | - Vahid Vahedian
- Department of Medical Laboratory Sciences, Faculty of MedicineIslamic Azad University Sari Iran
- Clinical Laboratory Medicine DepartmentRofeydeh Hospital University of Social Welfare and Rehabilitation Science Tehran Iran
| | - Saeed Nazari Soltan Ahmad
- Department of Biochemistry and Clinical Laboratories, Faculty of MedicineTabriz University of Medical Sciences Tabriz Iran
| | - Amir Zarebkohan
- Department of Medical Nanotechnology, Faculty of Advanced Medical SciencesTabriz University of Medical Sciences Tabriz Iran
| | | | - Vahid Hosseini
- Department of Biochemistry and Clinical Laboratories, Faculty of MedicineTabriz University of Medical Sciences Tabriz Iran
- Tuberculosis and Lung Disease Research CenterTabriz University of Medical Sciences Tabriz Iran
| | - Reza Rahbarghazi
- Tuberculosis and Lung Disease Research CenterTabriz University of Medical Sciences Tabriz Iran
- Department of Applied Cell Sciences, Faculty of Advanced Medical SciencesTabriz University of Medical Sciences Tabriz Iran
| |
Collapse
|
|
39
|
Koguchi M, Nakahara Y, Ito H, Wakamiya T, Yoshioka F, Ogata A, Inoue K, Masuoka J, Izumi H, Abe T. BMP4 induces asymmetric cell division in human glioma stem-like cells. Oncol Lett 2019; 19:1247-1254. [PMID: 31966054 PMCID: PMC6956386 DOI: 10.3892/ol.2019.11231] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Accepted: 11/25/2019] [Indexed: 12/14/2022] Open
Abstract
Glioblastoma (GBM) is a malignant tumor with a high recurrence rate and has very poor prognosis in humans. The median survival is still <2 years. Therefore, a new treatment strategy should be established. Recently, this cancer has been thought to be heterogeneous, consisting of cancer stem cells (CSCs) that are self-renewable, multipotent, and treatment resistant. So various strategies targeting glioma stem-like cells (GSCs) have been investigated. This study focused on strategies targeting GSCs through the induction of differentiation using bone morphogenetic protein 4 (BMP4). The expression of CD133, a cancer stem cell marker, under BMP4 treatment in GSCs was examined using flow cytometry, western blotting, and quantitative PCR. Immunofluorescent staining of GSCs was also performed to examine the type of cell division: asymmetric cell division (ACD) or symmetric cell division (SCD). We obtained the following results. The BMP4 treatment caused downregulation of CD133 expression. Moreover, it induced ACD in GSCs. While the ACD ratio was 23% without BMP4 treatment, it was 38% with BMP4 treatment (P=0.004). Furthermore, the tumor sphere assay demonstrated that BMP4 suppresses self-renewal ability. In conclusion, these findings may provide a new perspective on how BMP4 treatment reduces the tumorigenicity of GSCs.
Collapse
Affiliation(s)
- Motofumi Koguchi
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Yukiko Nakahara
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Hiroshi Ito
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Tomihiro Wakamiya
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Fumitaka Yoshioka
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Atsushi Ogata
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Kohei Inoue
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Jun Masuoka
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| | - Hideki Izumi
- Laboratory of Molecular Medicine, Life Sciences Institute, Saga Medical Center KOSEIKAN, Saga 840-8571, Japan
| | - Tatsuya Abe
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan
| |
Collapse
|
|
40
|
Wang Z, Xu Z, Jing G, Wang Q, Yang L, He X, Lin L, Niu J, Yang L, Li K, Liu Z, Qian Y, Wang S, Zhu R. Layered double hydroxide eliminate embryotoxicity of chemotherapeutic drug through BMP-SMAD signaling pathway. Biomaterials 2019; 230:119602. [PMID: 31735448 DOI: 10.1016/j.biomaterials.2019.119602] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Revised: 11/01/2019] [Accepted: 11/03/2019] [Indexed: 12/20/2022]
Abstract
Recent studies indicate that exogenous chemotherapy agents can cross the placenta barrier and cause fetal toxicity, while there exists barely alternative therapy for pregnant cancer patients. Here, we show a robust protective effect of layered double hydroxide (LDH) against etoposide (VP16) induced in vitro mouse embryonic stem cells (mESCs) toxicity and in vivo embryo developmental disorders. The nano-composite system (L-V) abrogated the original VP16 generated mitochondrial mediated mESCs toxicity totally, surprisingly maintained the pluripotency without leukemia inhibitory factor (LIF) and prevented the down-regulation of ectoderm marker expression during spontaneous embryoid bodies differentiation. Fetal growth retardation, the related placenta and skeletal structural abnormalities and long-term toxicity in the offspring were generated when pregnant mice exposed to VP16, while these detrimental effects were abolished when substituted with L-V. The different uterine drug accumulation of VP16 and L-V contributed to partly cause for the functional variation. And further transcriptome analysis confirmed developmental related BMP4-SMAD6 signaling pathway is of crucial importance. Our study revealed the devastating effects of VP16 on embryonic development and the toxicity-relieve method using nano-carrier system, which will provide important guidance for clinical application of LDH as alternative therapeutic system with minimal side effects for pregnant women diagnosed with cancer.
Collapse
Affiliation(s)
- Zhaojie Wang
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China; Division of Spine, Department of Orthopedics, Tongji Hospital Affiliated to Tongji University, School of Medicine, Shanghai, People's Republic of China; Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration, Tongji University, Ministry of Education, People's Republic of China
| | - Ziping Xu
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Guoxin Jing
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Qingxiu Wang
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Li Yang
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Xiaolie He
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Lijuan Lin
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Jintong Niu
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Linnan Yang
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Kun Li
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Zhongmin Liu
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China
| | - Yechang Qian
- Department of Respiratory Disease, Baoshan District Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, People's Republic of China.
| | - Shilong Wang
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China.
| | - Rongrong Zhu
- Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, People's Republic of China; Division of Spine, Department of Orthopedics, Tongji Hospital Affiliated to Tongji University, School of Medicine, Shanghai, People's Republic of China; Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration, Tongji University, Ministry of Education, People's Republic of China.
| |
Collapse
|
|
41
|
Mihajlović J, Diehl LAM, Hochhaus A, Clement JH. Inhibition of bone morphogenetic protein signaling reduces viability, growth and migratory potential of non-small cell lung carcinoma cells. J Cancer Res Clin Oncol 2019; 145:2675-2687. [PMID: 31531741 DOI: 10.1007/s00432-019-03026-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Accepted: 09/11/2019] [Indexed: 12/11/2022]
Abstract
PURPOSE BMP signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. A more in-depth analysis of lung cancer subtypes is needed to identify BMP-related therapeutic targets. We sought to examine the influence of BMP signaling on the viability, growth and migration properties of the cell line LCLC-103H, which originates from a large cell lung carcinoma with giant cells and an extended aneuploidy. METHODS We used BMP-4 and LDN-214117 as agonist/antagonist system for the BMP receptor type I signaling. Using flow cytometry, wound healing assay, trans-well assay and spheroid culture, we examined the influence of BMP signaling on cell viability, growth and migration. Molecular mechanisms underlying observed changes in cell migration were investigated via gene expression analysis of epithelial-mesenchymal transition (EMT) markers. RESULTS BMP signaling inhibition resulted in LCLC-103H cell apoptosis and necrosis 72 h after LDN-214117 treatment. Cell growth and proliferation are markedly affected by BMP signaling inhibition. Chemotactic motility and migratory ability of LCLC-103H cells were clearly hampered by LDN-214117 treatment. Cell migration changes after BMP signaling inhibition were shown to be coupled with considerable down-regulation of transcription factors involved in EMT, especially Snail. CONCLUSIONS BMP signaling inhibition in LCLC-103H cells leads to reduced growth and proliferation, hindered migration and accelerated cell death. The findings contribute to the pool of evidence on BMP signaling in lung cancer with a possibility of introducing BMP signaling inhibition as a novel therapeutic approach for the disease.
Collapse
Affiliation(s)
- Jelena Mihajlović
- Klinik Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Am Klinikum 1, 07747, Jena, Germany
| | - Laura A M Diehl
- Klinik Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Am Klinikum 1, 07747, Jena, Germany
| | - Andreas Hochhaus
- Klinik Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Am Klinikum 1, 07747, Jena, Germany
| | - Joachim H Clement
- Klinik Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Am Klinikum 1, 07747, Jena, Germany.
| |
Collapse
|
|
42
|
Valer JA, Sánchez-de-Diego C, Pimenta-Lopes C, Rosa JL, Ventura F. ACVR1 Function in Health and Disease. Cells 2019; 8:cells8111366. [PMID: 31683698 PMCID: PMC6912516 DOI: 10.3390/cells8111366] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Revised: 10/28/2019] [Accepted: 10/30/2019] [Indexed: 12/12/2022] Open
Abstract
Activin A receptor type I (ACVR1) encodes for a bone morphogenetic protein type I receptor of the TGFβ receptor superfamily. It is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterised by progressive heterotopic ossification. ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG), a malignant childhood brainstem glioma, and its function is being studied in other cancer types. Here, we review ACVR1 receptor function and signalling in physiological and pathological processes and its regulation according to cell type and mutational status. Learning from different functions and alterations linked to ACVR1 is a key step in the development of interdisciplinary research towards the identification of novel treatments for these pathologies.
Collapse
Affiliation(s)
- José Antonio Valer
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
| | - Cristina Sánchez-de-Diego
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
| | - Carolina Pimenta-Lopes
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
| | - Jose Luis Rosa
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
| | - Francesc Ventura
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
| |
Collapse
|
|
43
|
Dituri F, Cossu C, Mancarella S, Giannelli G. The Interactivity between TGFβ and BMP Signaling in Organogenesis, Fibrosis, and Cancer. Cells 2019; 8:E1130. [PMID: 31547567 PMCID: PMC6829314 DOI: 10.3390/cells8101130] [Citation(s) in RCA: 87] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Revised: 09/16/2019] [Accepted: 09/17/2019] [Indexed: 12/12/2022] Open
Abstract
The Transforming Growth Factor beta (TGFβ) and Bone Morphogenic Protein (BMP) pathways intersect at multiple signaling hubs and cooperatively or counteractively participate to bring about cellular processes which are critical not only for tissue morphogenesis and organogenesis during development, but also for adult tissue homeostasis. The proper functioning of the TGFβ/BMP pathway depends on its communication with other signaling pathways and any deregulation leads to developmental defects or diseases, including fibrosis and cancer. In this review we explore the cellular and physio-pathological contexts in which the synergism or antagonism between the TGFβ and BMP pathways are crucial determinants for the normal developmental processes, as well as the progression of fibrosis and malignancies.
Collapse
Affiliation(s)
- Francesco Dituri
- National Institute of Gastroenterology "S. De Bellis", Research Hospital, Castellana Grotte, 70013 Bari, Italy.
| | - Carla Cossu
- National Institute of Gastroenterology "S. De Bellis", Research Hospital, Castellana Grotte, 70013 Bari, Italy.
| | - Serena Mancarella
- National Institute of Gastroenterology "S. De Bellis", Research Hospital, Castellana Grotte, 70013 Bari, Italy.
| | - Gianluigi Giannelli
- National Institute of Gastroenterology "S. De Bellis", Research Hospital, Castellana Grotte, 70013 Bari, Italy.
| |
Collapse
|
|
44
|
Zhang F, Ma D, Liu T, Liu YH, Guo J, Song J, Wu Q, Pan Y, Zhang Y, Guo C, Teng C, Jin L. Expansion and Maintenance of CD133-Expressing Pancreatic Ductal Epithelial Cells by Inhibition of TGF-β Signaling. Stem Cells Dev 2019; 28:1236-1252. [PMID: 31311463 DOI: 10.1089/scd.2019.0087] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Restoring β-cell mass by the transplantation of pancreatic islets is an effective diabetes treatment, but it is limited by the shortage of donor organs. CD133-expressing pancreatic ductal epithelial cells (PDECs) have the ability to generate insulin-producing cells. The expansion of these cells is dependent on extrinsic niche factors, but few of those signals have been identified. In this study, CD133-expressing PDECs were purified by sorting from adult wild-type C57BL/6 mice and TGFβRIInull/null mice. Furthermore, using immunofluorescence and transplantation assays, we found that the inhibition of the transforming growth factor-β (TGF-β) pathway promoted the expansion of CD133-expressing PDECs for many generations and maintained the ability of CD133-expressing PDECs to generate insulin-producing cells. Moreover, western blot, qRT-PCR, and dual luciferase assays using TGF-β inhibitors were performed to identify the mechanisms by which TGF-β signaling regulates proliferation and differentiation. The results showed that the inhibition of TGF-β signaling enhanced Id2 binding to the promoter region of the cell proliferation repressor p16 and promoted the expansion of CD133-expressing PDECs, and the increased Id2 binding to NeuroD1 decreased the transcription of Pax6 to maintain CD133-expressing PDECs in the Pdx1-expression stage. Taken together, the effect of TGF-β antagonists on CD133-expressing PDECs reveals a novel paradigm of signaling that explains the balance between the expansion and differentiation of pancreatic duct epithelial progenitors.
Collapse
Affiliation(s)
- Fangfang Zhang
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Dongshen Ma
- Department of Pathology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, China
| | - Tingsheng Liu
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Yu Hong Liu
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Jiamin Guo
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Jing Song
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Qiong Wu
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Yi Pan
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Yanfeng Zhang
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Changying Guo
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| | - Chunbo Teng
- College of Life Science, Northeast Forestry University, Harbin, China
| | - Liang Jin
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, School of life Science and Technology, China Pharmaceutical University, Nanjing, People's Republic of China
| |
Collapse
|
|
45
|
Park S, Han SH, Kim HG, Jeong J, Choi M, Kim HY, Kim MG, Park JK, Han JE, Cho GJ, Kim MO, Ryoo ZY, Choi SK. Suppression of PRPF4 regulates pluripotency, proliferation, and differentiation in mouse embryonic stem cells. Cell Biochem Funct 2019; 37:608-617. [PMID: 31502671 DOI: 10.1002/cbf.3437] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2019] [Revised: 08/17/2019] [Accepted: 08/26/2019] [Indexed: 01/03/2023]
Abstract
Mouse embryonic stem cells (mESCs) are characterized by their self-renewal and pluripotency and are capable of differentiating into all three germ layers. For this reason, mESCs are considered a very important model for stem cell research and clinical applications in regenerative medicine. The pre-mRNA processing factor 4 (PRPF4) gene is known to have a major effect on pre-mRNA splicing and is also known to affect tissue differentiation during development. In this study, we investigated the effects of PRPF4 knockdown on mESCs. First, we allowed mESCs to differentiate naturally and observed a significant decrease in PRPF4 expression during the differentiation process. We then artificially induced the knockdown of PRPF4 in mESCs and observed the changes in the phenotype. When PRPF4 was knocked down, various genes involved in mESC pluripotency showed significantly decreased expression. In addition, mESC proliferation increased abnormally, accompanied by a significant increase in mESC colony size. The formation of mESC embryoid bodies and teratomas was delayed following PRPF4 knockdown. Based on these results, the reduced expression of PRPF4 affects mESC phenotypes and is a key factor in mESC. SIGNIFICANCE OF THE STUDY: Our results indicate that PRPF4 affects the properties of mESCs. Suppression of PRPF4 resulted in a decrease in pluripotency of mESC and promoted proliferation. In addition, suppression of PRPF4 also resulted in decreased apoptosis. Moreover, the inhibition of PRPF4 reduced the ability to differentiate and formation of teratoma in mESC. Our results demonstrated that PRPF4 is a key factor of controlling mESC abilities.
Collapse
Affiliation(s)
- Song Park
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea
| | - Se-Hyeon Han
- Department of News-team, SBS (Seoul Broadcasting Station), Yangchungu, Seoul, South Korea.,School of Media Communication, Hanyang University, Seongdonggu, Seoul, South Korea
| | - Hyeon-Gyeom Kim
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea.,School of Life Science, BK21 Plus KNU Creative Bioresearch Group, Kyungpook National University, Daegu, Korea
| | - Jain Jeong
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
| | - Minjee Choi
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea.,School of Life Science, BK21 Plus KNU Creative Bioresearch Group, Kyungpook National University, Daegu, Korea
| | - Hee-Yeon Kim
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea
| | - Min-Gi Kim
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea
| | - Jin-Kyu Park
- College of Veterinary Medicine, Kyungpook National University, Daegu, Korea
| | - Jee Eun Han
- College of Veterinary Medicine, Kyungpook National University, Daegu, Korea
| | - Gil-Jae Cho
- College of Veterinary Medicine, Kyungpook National University, Daegu, Korea
| | - Myoung Ok Kim
- Department of Animal Science, College of Ecology and Environment Science, Kyungpook National University, Sangju, Korea
| | - Zae Young Ryoo
- School of Life Science, BK21 Plus KNU Creative Bioresearch Group, Kyungpook National University, Daegu, Korea
| | - Seong-Kyoon Choi
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea.,Division of Biotechnology, DGIST, Daegu, Republic of Korea
| |
Collapse
|
|
46
|
Dupont G, Yilmaz E, Loukas M, Macchi V, De Caro R, Tubbs RS. Human embryonic stem cells: Distinct molecular personalities and applications in regenerative medicine. Clin Anat 2018; 32:354-360. [PMID: 30521112 PMCID: PMC6850663 DOI: 10.1002/ca.23318] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Accepted: 12/02/2018] [Indexed: 12/11/2022]
Abstract
The field of stem cell biology is exciting because it provides researchers and clinicians with seemingly unlimited applications for treating many human diseases. Stem cells are a renewable source of pluripotent cells that can differentiate into nearly all human cell types. In this article we focus particularly on human embryonic stem (hES) cells, derived from the inner cell mass of the blastocyst and cultured for expansion while remaining undifferentiated, to explore their unique molecular personalities and clinical applications. The aim of this literature review is to reflect the interest in hES cells and to provide a resource for researchers and clinicians interested in the molecular characteristics of such cells. Clin. Anat. 32:354–360, 2019. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.
Collapse
Affiliation(s)
| | - Emre Yilmaz
- Seattle Science Foundation, Seattle, Washington
| | - Marios Loukas
- Department of Anatomical Sciences, St. George's University, Grenada, West Indies
| | - Veronica Macchi
- Department of Neuroscience, Anatomy Institute, University of Padova, Padova, Italy
| | - Raffaele De Caro
- Department of Neuroscience, Anatomy Institute, University of Padova, Padova, Italy
| | - R Shane Tubbs
- Seattle Science Foundation, Seattle, Washington.,Department of Anatomical Sciences, St. George's University, Grenada, West Indies
| |
Collapse
|
|
47
|
Hahn S, Kim MS, Choi SY, Jeong S, Jee J, Kim HK, Jeong SY, Shin H, Kim HS, Park JS, Yoo J. Leucine-rich repeat-containing G-protein coupled receptor 5 enriched organoids under chemically-defined growth conditions. Biochem Biophys Res Commun 2018; 508:430-439. [PMID: 30503340 DOI: 10.1016/j.bbrc.2018.11.003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Accepted: 11/02/2018] [Indexed: 12/15/2022]
Abstract
An organoid is a complex, multi-cell three-dimensional (3D) structure that contains tissue-specific cells. Epithelial stem cells, which are marked by leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), have the potential for self-renewal and expansion as organoids. However, in the case of intestinal organoids from Lgr5-EGFP-IRES-CreERT2 transgenic mice, in vitro expansion of the Lgr5 expression is limited in a culture condition supplemented with essential proteins, such as epidermal growth factor (E), noggin (N), and R-spondin 1 (R). In this study, we hypothesized that self-renewal of Lgr5+ stem cells in a 3D culture system can be stimulated by defined compounds (CHIR99021, Valproic acid, Y-27632, and A83-01). Our results demonstrated that dissociated single cells from organoids were organized into a 3D structure in the four compounds containing the ENR culture medium in a 3D and two-dimensional (2D) culture system. Moreover, the Lgr5 expression level of organoids from the ENR- and compound-containing media increased. Furthermore, the conversion of cultured Lgr5+ stem cells from 2D to 3D was confirmed. Therefore, defined compounds promote the expansion of Lgr5+ stem cells in organoids.
Collapse
Affiliation(s)
- Soojung Hahn
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - Mi Sun Kim
- Department of Radiation Oncology, Bundang CHA Medical Center, South Korea
| | - Seon Young Choi
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - Sukin Jeong
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - JooHyun Jee
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - Han Kyung Kim
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - Sang Yun Jeong
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea
| | - Hyunsoo Shin
- Department of Radiation Oncology, Bundang CHA Medical Center, South Korea
| | - Hyoung Sun Kim
- Pancreatobiliary Cancer Clinic, Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, South Korea
| | - Joon Seong Park
- Pancreatobiliary Cancer Clinic, Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, South Korea; Department of Surgery, Gangnam Severance Hospital, South Korea
| | - Jongman Yoo
- Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea.
| |
Collapse
|
|
48
|
Azami T, Matsumoto K, Jeon H, Waku T, Muratani M, Niwa H, Takahashi S, Ema M. Klf5 suppresses ERK signaling in mouse pluripotent stem cells. PLoS One 2018; 13:e0207321. [PMID: 30452437 PMCID: PMC6242311 DOI: 10.1371/journal.pone.0207321] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Accepted: 10/29/2018] [Indexed: 01/05/2023] Open
Abstract
Mouse embryonic stem cells (ESCs) are pluripotent stem cells, which have the ability to differentiate into all three germ layers: mesoderm, endoderm, and ectoderm. Proper levels of phosphorylated extracellular signal-regulated kinase (pERK) are critical for maintaining pluripotency, as elevated pERK evoked by fibroblast growth factor (FGF) receptor activation results in differentiation of ESCs, while, conversely, reduction of pERK by a MEK inhibitor maintains a pluripotent ground state. However, mechanisms underlying proper control of pERK levels in mouse ESCs are not fully understood. Here, we find that Klf5, a Krüppel-like transcription factor family member, is a component of pERK regulation in mouse ESCs. We show that ERK signaling is overactivated in Klf5-KO ESCs and the overactivated ERK in Klf5-KO ESCs is suppressed by the introduction of Klf5, but not Klf2 or Klf4, indicating a unique role for Klf5 in ERK suppression. Moreover, Klf5 regulates Spred1, a negative regulator of the FGF-ERK pathway. Klf5 also facilitates reprogramming of EpiSCs into a naïve state in combination with a glycogen synthase kinase 3 inhibitor and LIF, and in place of a MEK inhibitor. Taken together, these results show for the first time that Klf5 has a unique role suppressing ERK activity in mouse ESCs.
Collapse
Affiliation(s)
- Takuya Azami
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga, Japan
| | - Ken Matsumoto
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga, Japan
| | - Hyojung Jeon
- Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Tsuyoshi Waku
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
| | - Masafumi Muratani
- Department of Genome Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Hitoshi Niwa
- Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan
| | - Satoru Takahashi
- Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
- International Institute for Integrative Sleep Medicine (IIIS), University of Tsukuba, Tsukuba, Ibaraki, Japan
- Life Science Center (TARA), University of Tsukuba, Tsukuba, Ibaraki, Japan
- Transborder Medical Research Center (TMRC), University of Tsukuba, Tsukuba, Ibaraki, Japan
- Laboratory Animal Resource Center (LARC), University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Masatsugu Ema
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga, Japan
- PRESTO, Japan Science and Technology Agency, Saitama, Japan
- * E-mail:
| |
Collapse
|
|
49
|
Teramura T, Matsuda K, Takehara T, Shinohara K, Miyashita Y, Mieno Y, Mori T, Fukuda K, Suzuki K, Suemori H. Laser-assisted cell removing (LACR) technology contributes to the purification process of the undifferentiated cell fraction during pluripotent stem cell culture. Biochem Biophys Res Commun 2018; 503:3114-3120. [PMID: 30143262 DOI: 10.1016/j.bbrc.2018.08.101] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2018] [Accepted: 08/14/2018] [Indexed: 10/28/2022]
Abstract
Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine.
Collapse
Affiliation(s)
- Takeshi Teramura
- Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, Osaka, Japan; Department of Obstetrics and Gynecology, Mie University Graduate School of Medicine, Japan.
| | | | - Toshiyuki Takehara
- Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, Osaka, Japan
| | | | | | | | - Tatsufumi Mori
- Kindai University Life Science Research Institute, Kindai University, Japan
| | - Kanji Fukuda
- Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, Osaka, Japan
| | | | - Hirofumi Suemori
- Laboratory of Embryonic Stem Cell Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
| |
Collapse
|
|
50
|
Bernardo AS, Jouneau A, Marks H, Kensche P, Kobolak J, Freude K, Hall V, Feher A, Polgar Z, Sartori C, Bock I, Louet C, Faial T, Kerstens HHD, Bouissou C, Parsonage G, Mashayekhi K, Smith JC, Lazzari G, Hyttel P, Stunnenberg HG, Huynen M, Pedersen RA, Dinnyes A. Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state. Biol Open 2018; 7:bio.033282. [PMID: 30026265 PMCID: PMC6124576 DOI: 10.1242/bio.033282] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro. Amongst these are the pluripotency-linked genes Klf4 and Lin28b. The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human. Summary: Interspecies comparison of mouse, bovine and pig embryos revealed conserved genes which distinguish between naïve and primed pluripotency states, including in human. Some of these genes interfere with the pluripotency network and lead to differentiation.
Collapse
Affiliation(s)
- Andreia S Bernardo
- The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, UK .,Developmental Biology Department, The Francis Crick Institute, 1 Midland Rd, Kings Cross, London NW1 1AT, UK
| | - Alice Jouneau
- UMR BDR, INRA, ENVA, Université Paris Saclay, 78350, Jouy en Josas, France
| | - Hendrik Marks
- Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6500 HB Nijmegen, The Netherlands
| | - Philip Kensche
- Center for Molecular and Biomolecular Informatics, Radboud Institute of Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands
| | | | - Kristine Freude
- Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Groennegaardsvej 7, 1870 Frederiksberg C, Denmark
| | - Vanessa Hall
- Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Groennegaardsvej 7, 1870 Frederiksberg C, Denmark
| | - Anita Feher
- BioTalentum Ltd, Gödöllő, 2100 Godollo, Hungary
| | | | - Chiara Sartori
- Avantea, Laboratory of Reproductive Technologies, Cremona, 26100 Cremona, Italy.,Department of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland
| | - Istvan Bock
- BioTalentum Ltd, Gödöllő, 2100 Godollo, Hungary
| | - Claire Louet
- UMR BDR, INRA, ENVA, Université Paris Saclay, 78350, Jouy en Josas, France
| | - Tiago Faial
- The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, UK
| | - Hindrik H D Kerstens
- Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6500 HB Nijmegen, The Netherlands
| | - Camille Bouissou
- Developmental Biology Department, The Francis Crick Institute, 1 Midland Rd, Kings Cross, London NW1 1AT, UK
| | - Gregory Parsonage
- The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, UK.,Developmental Biology Department, The Francis Crick Institute, 1 Midland Rd, Kings Cross, London NW1 1AT, UK
| | - Kaveh Mashayekhi
- BioTalentum Ltd, Gödöllő, 2100 Godollo, Hungary.,Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Groennegaardsvej 7, 1870 Frederiksberg C, Denmark
| | - James C Smith
- Developmental Biology Department, The Francis Crick Institute, 1 Midland Rd, Kings Cross, London NW1 1AT, UK
| | - Giovanna Lazzari
- Avantea, Laboratory of Reproductive Technologies, Cremona, 26100 Cremona, Italy
| | - Poul Hyttel
- Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Groennegaardsvej 7, 1870 Frederiksberg C, Denmark
| | - Hendrik G Stunnenberg
- Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6500 HB Nijmegen, The Netherlands
| | - Martijn Huynen
- Center for Molecular and Biomolecular Informatics, Radboud Institute of Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands
| | - Roger A Pedersen
- The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, UK
| | - Andras Dinnyes
- BioTalentum Ltd, Gödöllő, 2100 Godollo, Hungary .,Molecular Animal Biotechnology Laboratory, Szent István University, H-2100 Godollo, Gödöllő, Hungary.,Departments of Equine Sciences and Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, The Netherlands
| |
Collapse
|