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Yu M, Qin K, Fan J, Zhao G, Zhao P, Zeng W, Chen C, Wang A, Wang Y, Zhong J, Zhu Y, Wagstaff W, Haydon RC, Luu HH, Ho S, Lee MJ, Strelzow J, Reid RR, He TC. The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities. Genes Dis 2024; 11:101026. [PMID: 38292186 PMCID: PMC10825312 DOI: 10.1016/j.gendis.2023.04.042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 03/18/2023] [Accepted: 04/12/2023] [Indexed: 02/01/2024] Open
Abstract
The evolutionarily conserved Wnt signaling pathway plays a central role in development and adult tissue homeostasis across species. Wnt proteins are secreted, lipid-modified signaling molecules that activate the canonical (β-catenin dependent) and non-canonical (β-catenin independent) Wnt signaling pathways. Cellular behaviors such as proliferation, differentiation, maturation, and proper body-axis specification are carried out by the canonical pathway, which is the best characterized of the known Wnt signaling paths. Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues. This includes but is not limited to embryonic, hematopoietic, mesenchymal, gut, neural, and epidermal stem cells. Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties. Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders. Not surprisingly, aberrant Wnt signaling is also associated with a wide variety of diseases, including cancer. Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation, epithelial-mesenchymal transition, and metastasis. Altogether, advances in the understanding of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway. Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt, this review aims to summarize the current knowledge of Wnt signaling in stem cells, aberrations to the Wnt pathway associated with diseases, and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.
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Affiliation(s)
- Michael Yu
- School of Medicine, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Kevin Qin
- School of Medicine, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Jiaming Fan
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Ministry of Education Key Laboratory of Diagnostic Medicine, and Department of Clinical Biochemistry, The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Guozhi Zhao
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
| | - Piao Zhao
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
| | - Wei Zeng
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Department of Neurology, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, Guangdong 523475, China
| | - Connie Chen
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Annie Wang
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Yonghui Wang
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Department of Clinical Laboratory Medicine, Shanghai Jiaotong University School of Medicine, Shanghai 200000, China
| | - Jiamin Zhong
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Ministry of Education Key Laboratory of Diagnostic Medicine, and Department of Clinical Biochemistry, The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Yi Zhu
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Department of Orthopaedic Surgery, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China
| | - William Wagstaff
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Rex C. Haydon
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Hue H. Luu
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Sherwin Ho
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Michael J. Lee
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Jason Strelzow
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Russell R. Reid
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Laboratory of Craniofacial Suture Biology and Development, Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
| | - Tong-Chuan He
- Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Laboratory of Craniofacial Suture Biology and Development, Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
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Chang Y, Syahirah R, Oprescu SN, Wang X, Jung J, Cooper SH, Torregrosa-Allen S, Elzey BD, Hsu AY, Randolph LN, Sun Y, Kuang S, Broxmeyer HE, Deng Q, Lian X, Bao X. Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells. Biomaterials 2022; 285:121569. [PMID: 35567999 PMCID: PMC10065832 DOI: 10.1016/j.biomaterials.2022.121569] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2021] [Revised: 04/23/2022] [Accepted: 05/03/2022] [Indexed: 12/17/2022]
Abstract
Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFβ inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.
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Affiliation(s)
- Yun Chang
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA; Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA
| | - Ramizah Syahirah
- Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA
| | - Stephanie N Oprescu
- Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA; Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA
| | - Xuepeng Wang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Juhyung Jung
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA; Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA
| | - Scott H Cooper
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | | | - Bennett D Elzey
- Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA; Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, 47907, USA
| | - Alan Y Hsu
- Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA; Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
| | - Lauren N Randolph
- Departments of Biomedical Engineering, Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA
| | - Yufei Sun
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA
| | - Shihuan Kuang
- Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA; Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA
| | - Hal E Broxmeyer
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Qing Deng
- Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA; Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA.
| | - Xiaojun Lian
- Departments of Biomedical Engineering, Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA.
| | - Xiaoping Bao
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA; Purdue University Center for Cancer Research, West Lafayette, IN, 47907, USA.
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Gautam DK, Chimata AV, Gutti RK, Paddibhatla I. Comparative hematopoiesis and signal transduction in model organisms. J Cell Physiol 2021; 236:5592-5619. [PMID: 33492678 DOI: 10.1002/jcp.30287] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Revised: 12/24/2020] [Accepted: 01/08/2021] [Indexed: 12/21/2022]
Abstract
Hematopoiesis is a continuous phenomenon involving the formation of hematopoietic stem cells (HSCs) giving rise to diverse functional blood cells. This developmental process of hematopoiesis is evolutionarily conserved, yet comparably different in various model organisms. Vertebrate HSCs give rise to all types of mature cells of both the myeloid and the lymphoid lineages sequentially colonizing in different anatomical tissues. Signal transduction in HSCs facilitates their potency and specifies branching of lineages. Understanding the hematopoietic signaling pathways is crucial to gain insights into their deregulation in several blood-related disorders. The focus of the review is on hematopoiesis corresponding to different model organisms and pivotal role of indispensable hematopoietic pathways. We summarize and discuss the fundamentals of blood formation in both invertebrate and vertebrates, examining the requirement of key signaling nexus in hematopoiesis. Knowledge obtained from such comparative studies associated with developmental dynamics of hematopoiesis is beneficial to explore the therapeutic options for hematopoietic diseases.
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Affiliation(s)
- Dushyant Kumar Gautam
- Department of Biochemistry, School of Life Sciences (SLS), University of Hyderabad, Hyderabad, Telangana, India
| | | | - Ravi Kumar Gutti
- Department of Biochemistry, School of Life Sciences (SLS), University of Hyderabad, Hyderabad, Telangana, India
| | - Indira Paddibhatla
- Department of Biochemistry, School of Life Sciences (SLS), University of Hyderabad, Hyderabad, Telangana, India
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Kundu MC, Gore LR, Maguire S, Gilmartin AG. Development and Characterization of a Model for Inducing Fetal Hemoglobin Production in Cynomolgus Macaques ( Macaca fasicularis). Comp Med 2018; 68:396-402. [PMID: 30092854 DOI: 10.30802/aalas-cm-17-000124] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Hydroxyurea induces production of fetal hemoglobin (HbF), a tetramer of α and γ globin proteins and corresponding heme molecules, normally found in less than 1% of adult RBC. Increases in circulating HbF are correlated with clinical improvement of patients with hemoglobinopathies, and hydroxyurea, as a daily medication, is the standard treatment for sickle cell anemia. Although olive baboons (Papio anubis) are considered a key model species for HbF induction, cynomolgus macaques (Macaca fasicularis) are another species that conserves the ability to produce HbF into maturity. In this study, moderate anemia was experimentally induced in cynomolgus macaques by phlebotomy, to stimulate accelerated erythropoiesis and HbF production. In contrast to previous studies, vascular access ports were implanted for phlebotomy of conscious monkeys, followed by fluid replacement. As total Hgb levels dropped, reticulocyte counts and the percentage of HbF-expressing cells increased. Once total Hgb levels declined to less than 8 g/dL, 2 courses of oral hydroxyurea (once daily for 5 d) were completed, with a 9-d interval between courses. After hydroxyurea dosing, the percentage of HbF-expressing cells and total HbF were increased significantly. In addition, a significant but transient decrease in reticulocyte count and a transient increase in MCV occurred, replicating the characteristic response of patients receiving hydroxyurea. Daily clinical observations revealed no serious health issues or decreases in food consumption or activity levels. Methods were established for assessing the patency of vascular access ports. This study details a new protocol for the safe and routine induction of moderate anemia in cynomolgus macaques and validates its use in the investigation of novel pharmacologic entities to induce the production of HbF.
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Affiliation(s)
- Mila C Kundu
- Integrated Biological Platform Sciences, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania, USA; Veterinary Sciences, Bristol-Myers Squibb, New Brunswick, New Jersey, USA
| | - Liz R Gore
- Haemoglobin DPU, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania, USA
| | - Sean Maguire
- Integrated Biological Platform Sciences, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania, USA.
| | - Aidan G Gilmartin
- Haemoglobin DPU, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania, USA
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5
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Natsumoto B, Shoda H, Fujio K, Otsu M, Yamamoto K. Investigation of the pathogenesis of autoimmune diseases by iPS cells. ACTA ACUST UNITED AC 2017; 40:48-53. [PMID: 28539554 DOI: 10.2177/jsci.40.48] [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] [Indexed: 11/15/2022]
Abstract
The pluripotent stem cells have a self-renewal ability and can be differentiated into theoretically all of cell types. The induced pluripotent stem (iPS) cells overcame the ethical problems of the human embryonic stem (ES) cell, and enable pathologic analysis of intractable diseases and drug discovery. The in vitro disease model using disease-specific iPS cells enables repeated analyses of human cells without influence of environment factors. Even though autoimmune diseases are polygenic diseases, autoimmune disease-specific iPS cells are thought to be a promising tool for analyzing the pathogenesis of the diseases and drug discovery in future.
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Affiliation(s)
- Bunki Natsumoto
- Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo
| | - Hirofumi Shoda
- Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo
| | - Keishi Fujio
- Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo
| | - Makoto Otsu
- Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, the University of Tokyo
| | - Kazuhiko Yamamoto
- Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo
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6
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D'Souza SS, Maufort J, Kumar A, Zhang J, Smuga-Otto K, Thomson JA, Slukvin II. GSK3β Inhibition Promotes Efficient Myeloid and Lymphoid Hematopoiesis from Non-human Primate-Induced Pluripotent Stem Cells. Stem Cell Reports 2016; 6:243-56. [PMID: 26805448 PMCID: PMC4750098 DOI: 10.1016/j.stemcr.2015.12.010] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2015] [Revised: 12/18/2015] [Accepted: 12/20/2015] [Indexed: 01/01/2023] Open
Abstract
Advances in the scalable production of blood cells from induced pluripotent stem cells (iPSCs) open prospects for the clinical translation of de novo generated blood products, and evoke the need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, the outcomes of cellular therapies in non-human primate (NHP) models can be readily extrapolated to a clinical setting. However, the use of this model is hampered by relatively low efficiency of blood generation and lack of lymphoid potential in NHP-iPSC differentiation cultures. Here, we generated transgene-free iPSCs from different NHP species and showed the efficient induction of mesoderm, myeloid, and lymphoid cells from these iPSCs using a GSK3β inhibitor. Overall, our studies enable scalable production of hematopoietic progenitors from NHP-iPSCs, and lay the foundation for preclinical testing of iPSC-based therapies for blood and immune system diseases in an NHP model.
Generation of transgene-free iPSCs from various non-human primate (NHP) species GSK3β inhibition is essential for induction of mesoderm and blood from NHP-iPSCs Efficient generation of CD34+CD45+CD90+CD38−CD45RA− progenitors from NHP-iPSCs Efficient T and NK cell production from NHP-iPSCs
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Affiliation(s)
- Saritha S D'Souza
- National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA
| | - John Maufort
- National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA
| | - Akhilesh Kumar
- National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA
| | - Jiuchun Zhang
- Morgridge Institute for Research, 309 North Orchard Street, Madison, WI 53715, USA
| | - Kimberley Smuga-Otto
- Morgridge Institute for Research, 309 North Orchard Street, Madison, WI 53715, USA
| | - James A Thomson
- Morgridge Institute for Research, 309 North Orchard Street, Madison, WI 53715, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53707, USA; Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, CA 93106, USA
| | - Igor I Slukvin
- National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53707, USA; Department of Pathology and Laboratory Medicine, University of Wisconsin, 1685 Highland Avenue, Madison WI 53705, USA.
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7
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Guye P, Ebrahimkhani MR, Kipniss N, Velazquez JJ, Schoenfeld E, Kiani S, Griffith LG, Weiss R. Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6. Nat Commun 2016; 7:10243. [PMID: 26732624 PMCID: PMC4729822 DOI: 10.1038/ncomms10243] [Citation(s) in RCA: 113] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Accepted: 11/20/2015] [Indexed: 01/15/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues. There has been limited success in generating tissues from human induced pluripotent stem cells (hiPSCs). Here, the authors genetically engineer expression of the transcription factor Gata6 in a single isogenic hiPSC population resulting in complex tissue structures that exhibit liver bud-like properties.
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Affiliation(s)
- Patrick Guye
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA.,Synthetic Biology Center, MIT, Cambridge, Massachusetts 02139, USA
| | - Mohammad R Ebrahimkhani
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA
| | - Nathan Kipniss
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA.,Synthetic Biology Center, MIT, Cambridge, Massachusetts 02139, USA
| | - Jeremy J Velazquez
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA
| | - Eldi Schoenfeld
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA.,Synthetic Biology Center, MIT, Cambridge, Massachusetts 02139, USA
| | - Samira Kiani
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,Synthetic Biology Center, MIT, Cambridge, Massachusetts 02139, USA
| | - Linda G Griffith
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA
| | - Ron Weiss
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.,MIT Emergent Behaviors of Integrated Cellular Systems (EBICS) Center, Cambridge, Massachusetts 02139, USA.,Synthetic Biology Center, MIT, Cambridge, Massachusetts 02139, USA
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8
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Pipino C, Mukherjee S, David AL, Blundell MP, Shaw SW, Sung P, Shangaris P, Waters JJ, Ellershaw D, Cavazzana M, Mostoslavsky G, Pandolfi A, Pierro A, Guillot PV, Thrasher AJ, De Coppi P. Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking. Cell Reprogram 2014; 16:331-44. [PMID: 25162836 DOI: 10.1089/cell.2013.0091] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular, hematological, and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2, OCT4, KLF-4, and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-), γ-chain(-/-), C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment, immunostaining, and RT-PCR. Additionally, the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery.
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Affiliation(s)
- Caterina Pipino
- 1 Surgery Unit, Institute of Child Health, University College London , London, WC1N 1EH, United Kingdom
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9
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Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions. PLoS One 2013; 8:e59243. [PMID: 23573196 PMCID: PMC3616072 DOI: 10.1371/journal.pone.0059243] [Citation(s) in RCA: 114] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2012] [Accepted: 02/13/2013] [Indexed: 01/12/2023] Open
Abstract
Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×106±0.3×106 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.
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10
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Ramesh B, Guhathakurta S. Large-scale in-vitro expansion of RBCs from hematopoietic stem cells. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2012; 41:42-51. [PMID: 22834784 DOI: 10.3109/10731199.2012.702315] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The quest for RBCs in transfusion medicine has prompted scientists to explore the large-scale expansion of human RBCs from various sources. The successful production of RBCs in the laboratory depends on the selection of potential cell source, optimized culture, bio-physiological parameters, clinically applicable culture media that yields a scalable, contamination-free, non-reactive, non-tumorogenic, stable and functional end product. The expansion protocol considering the in vivo factors involved in homeostasis can generate a cost-effective and readily available cell source for transfusion. This review paper discusses several approaches used to expand RBCs from various sources of stem cells.
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Affiliation(s)
- Balasundari Ramesh
- Department of Stem Cells and Tissue Engineering, Frontier Life Line Pvt Ltd., Mugappair, Chennai, India
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11
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Ebihara Y, Ma F, Tsuji K. Generation of red blood cells from human embryonic/induced pluripotent stem cells for blood transfusion. Int J Hematol 2012; 95:610-6. [PMID: 22648827 DOI: 10.1007/s12185-012-1107-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2012] [Revised: 05/14/2012] [Accepted: 05/14/2012] [Indexed: 12/19/2022]
Abstract
Red blood cell (RBC) transfusion is necessary for many patients with emergency or hematological disorders. However, to date the supply of RBCs remains labile and dependent on voluntary donations. In addition, the transmission of infectious disease via blood transfusion from unspecified donors remains a risk. Establishing a large quantity of safe RBCs would help to address this issue. Human embryonic stem (hES) cells and the recently established human induced pluripotent stem (hiPS) cells represent potentially unlimited sources of donor-free RBCs for blood transfusion, as they can proliferate indefinitely in vitro. Extensive research has been done to efficiently generate transfusable RBCs from hES/iPS cells. Nevertheless, a number of challenges must be overcome before the clinical usage of hES/iPS cell-derived RBCs can become a reality.
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Affiliation(s)
- Yasuhiro Ebihara
- Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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12
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Saito MK, Niwa A. Disease-associated iPS cell lines representing hematological and immunological disorders. Inflamm Regen 2012. [DOI: 10.2492/inflammregen.32.171] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
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13
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Permanent expression of midbrain dopaminergic neurons traits in differentiated amniotic epithelial cells. Neurosci Lett 2012; 506:22-7. [DOI: 10.1016/j.neulet.2011.10.038] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2011] [Revised: 09/27/2011] [Accepted: 10/14/2011] [Indexed: 11/18/2022]
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14
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Sharma AK, Bury MI, Marks AJ, Fuller NJ, Meisner JW, Tapaskar N, Halliday LC, Matoka DJ, Cheng EY. A nonhuman primate model for urinary bladder regeneration using autologous sources of bone marrow-derived mesenchymal stem cells. Stem Cells 2011; 29:241-50. [PMID: 21732482 DOI: 10.1002/stem.568] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Animal models that have been used to examine the regenerative capacity of cell-seeded scaffolds in a urinary bladder augmentation model have ultimately translated poorly in the clinical setting. This may be due to a number of factors including cell types used for regeneration and anatomical/physiological differences between lower primate species and their human counterparts. We postulated that mesenchymal stem cells (MSCs) could provide a cell source for partial bladder regeneration in a newly described nonhuman primate bladder (baboon) augmentation model. Cell-sorted CD105(+) /CD73(+) /CD34(-) /CD45(-) baboon MSCs transduced with green fluorescent protein (GFP) were seeded onto small intestinal submucosa (SIS) scaffolds. Baboons underwent an approximate 40%-50% cystectomy followed by augmentation cystoplasty with the aforementioned scaffolds or controls and finally enveloped with omentum. Bladders from sham, unseeded SIS, and MSC/SIS scaffolds were subjected to trichrome, H&E, and immunofluorescent staining 10 weeks postaugmentation. Immunofluorescence staining for muscle markers combined with an anti-GFP antibody revealed that >90% of the cells were GFP(+) /muscle marker(+) and >70% were GFP(+) /Ki-67(+) demonstrating grafted cells were present and actively proliferating within the grafted region. Trichrome staining of MSC/SIS-augmented bladders exhibited typical bladder architecture and quantitative morphometry analyses revealed an approximate 32% and 52% muscle to collagen ratio in unseeded versus seeded animals, respectively. H&E staining revealed a lack of infiltration of inflammatory cells in grafted animals and in corresponding kidneys and ureters. Simple cystometry indicated recovery between 28% and 40% of native bladder capacity. Data demonstrate MSC/SIS composites support regeneration of bladder tissue and validate this new bladder augmentation model.
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Affiliation(s)
- Arun K Sharma
- Division of Pediatric Urology; Children's Memorial Hospital of Chicago, Chicago, Illinois, USA.
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15
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Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells. PLoS One 2011; 6:e25761. [PMID: 22022444 PMCID: PMC3192723 DOI: 10.1371/journal.pone.0025761] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2011] [Accepted: 09/12/2011] [Indexed: 12/20/2022] Open
Abstract
We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.
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16
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Niwa A, Heike T, Umeda K, Oshima K, Kato I, Sakai H, Suemori H, Nakahata T, Saito MK. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors. PLoS One 2011; 6:e22261. [PMID: 21818303 PMCID: PMC3144871 DOI: 10.1371/journal.pone.0022261] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2011] [Accepted: 06/18/2011] [Indexed: 12/16/2022] Open
Abstract
Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.
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Affiliation(s)
- Akira Niwa
- Clinical Application Department, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toshio Heike
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Katsutsugu Umeda
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas, United States of America
| | - Koichi Oshima
- Clinical Application Department, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Itaru Kato
- Clinical Application Department, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Hiromi Sakai
- Waseda Bioscience Research Institute in Helios, Singapore
| | - Hirofumi Suemori
- Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Tatsutoshi Nakahata
- Clinical Application Department, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Megumu K. Saito
- Clinical Application Department, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- * E-mail:
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17
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Hiroyama T, Miharada K, Kurita R, Nakamura Y. Plasticity of cells and ex vivo production of red blood cells. Stem Cells Int 2011; 2011:195780. [PMID: 21785608 PMCID: PMC3137953 DOI: 10.4061/2011/195780] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2011] [Accepted: 05/13/2011] [Indexed: 11/21/2022] Open
Abstract
The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If transfusable RBCs could be produced abundantly from certain resources, it would be very useful. Our group has developed a method to produce enucleated RBCs efficiently from hematopoietic stem/progenitor cells present in umbilical cord blood. More recently, it was reported that enucleated RBCs could be abundantly produced from human embryonic stem (ES) cells. The common obstacle for application of these methods is that they require very high cost to produce sufficient number of RBCs that are applicable in the clinic. If erythroid cell lines (immortalized cell lines) able to produce transfusable RBCs ex vivo were established, they would be valuable resources. Our group developed a robust method to obtain immortalized erythroid cell lines able to produce mature RBCs. To the best of our knowledge, this was the first paper to show the feasibility of establishing immortalized erythroid progenitor cell lines able to produce enucleated RBCs ex vivo. This result strongly suggests that immortalized human erythroid progenitor cell lines able to produce mature RBCs ex vivo can also be established.
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Affiliation(s)
- Takashi Hiroyama
- Cell Engineering Division, RIKEN BioResource Center, Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074, Japan
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18
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Morishima T, Watanabe KI, Niwa A, Fujino H, Matsubara H, Adachi S, Suemori H, Nakahata T, Heike T. Neutrophil differentiation from human-induced pluripotent stem cells. J Cell Physiol 2011; 226:1283-91. [PMID: 20945397 DOI: 10.1002/jcp.22456] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Induced pluripotent stem (iPS) cells are of potential value not only for regenerative medicine, but also for disease investigation. The present study describes the development of a neutrophil differentiation system from human iPS cells (hiPSCs) and the analysis of neutrophil function and differentiation. The culture system used consisted of the transfer of hiPSCs onto OP9 cells and their culture with vascular endothelial growth factor (VEGF). After 10 days, TRA 1-85(+) CD34(+) VEGF receptor-2 (VEGFR-2)(high) cells were sorted and co-cultured with OP9 cells in the presence of hematopoietic cytokines for 30 days. Floating cells were collected and subjected to morphological and functional analysis. These hiPSC-derived neutrophils were similar to peripheral blood mature neutrophils in morphology, contained functional neutrophil specific granules, and were equipped with the basic functions such as phagocytosis, superoxide production, and chemotaxis. In the process of differentiation, myeloid cells appeared sequentially from immature myeloblasts to mature segmented neutrophils. Expression patterns of surface antigen, transcription factors, and granule proteins during differentiation were also similar to those of granulopoiesis in normal bone marrow. In conclusion, differentiation of mature neutrophils from hiPSCs was successfully induced in a similar process to normal granulopoiesis using an OP9 co-culture system. This system may be applied to elucidate the pathogenesis of various hematological diseases that affect neutrophils.
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Affiliation(s)
- Tatsuya Morishima
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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19
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Tsuchiya A, Kanefuji T, Suda T, Aoyagi T, Osaki A, Togashi T, Kawauchi Y, Fushiki M, Watanabe G, Hirota M, Nomoto M, Ajioka Y, Aoyagi Y. Alpha-fetoprotein-producing adenocarcinoma in which the metastatic route was determined from calcified lesions. Clin J Gastroenterol 2011; 4:89-94. [PMID: 26190712 DOI: 10.1007/s12328-011-0209-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2010] [Accepted: 01/24/2011] [Indexed: 02/07/2023]
Abstract
Alpha-fetoprotein (AFP)-producing adenocarcinoma is known for its rapid progression and poor prognosis, and chemotherapy regimens are yet to be standardized. Here we describe the first report of AFP-producing adenocarcinoma with calcification. The metastatic route was visualized from the calcification, and combination chemotherapy was performed. A 77-year-old Japanese man was transferred to our hospital for treatment of liver tumors. Computed tomography (CT) revealed multiple liver tumors with portal vein tumor thrombosis. The tumors were highly attenuated before enhancement, suggesting various degrees of calcification. Serum levels of carcinoembryonic antigen (CEA), AFP, and the proportion of fucosylated AFP were considerably elevated. Gastroduodenoscopy revealed an elevated tumor occupying the duodenal bulb with an ulcerative lesion in the vicinity of the gastroduodenal junction, and biopsy specimens from the duodenum and liver revealed medullary adenocarcinoma with calcification. Three-dimensional reconstruction of CT images clearly showed that the calcified lesions had spread from the gastroduodenal junction to the liver via a route comprising the corresponding local vein, the superior mesenteric vein, and portal vein. The patient was accordingly diagnosed with calcified AFP-producing adenocarcinoma with multiple liver metastases. Combination chemotherapy using TS-1 and cisplatin (CDDP) resulted in a striking response for the initial 4 months in terms of tumor markers and CT findings. This is the first report of AFP-producing adenocarcinoma with calcification. A metastatic route from the primary tumor to the liver was clearly visualized by tracing the calcified lesions. Combination chemotherapy based on 5-fluorouracil and CDDP may have the potential to prolong survival.
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Affiliation(s)
- Atsunori Tsuchiya
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan.
| | - Tsutomu Kanefuji
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Takeshi Suda
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Tomoya Aoyagi
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Akihiko Osaki
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Tadayuki Togashi
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Yusuke Kawauchi
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Mae Fushiki
- Division of Molecular Diagnostic Pathology, Niigata University Medical and Dental Hospital, Niigata, Japan
| | - Gen Watanabe
- Division of Molecular Diagnostic Pathology, Niigata University Medical and Dental Hospital, Niigata, Japan
| | - Masaki Hirota
- Department of Surgery, Niigata Prefecture Muikamachi Hospital, Minamiuonuma, Japan
| | - Minoru Nomoto
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Yoichi Ajioka
- Division of Molecular Diagnostic Pathology, Niigata University Medical and Dental Hospital, Niigata, Japan
| | - Yutaka Aoyagi
- Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, 1-757 Asahimachi-Dori, Chuo-ku, Niigata, 951-8510, Japan
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20
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Wianny F, Bourillot PY, Dehay C. Embryonic stem cells in non-human primates: An overview of neural differentiation potential. Differentiation 2011; 81:142-52. [PMID: 21296479 DOI: 10.1016/j.diff.2011.01.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2010] [Revised: 12/18/2010] [Accepted: 01/11/2011] [Indexed: 12/11/2022]
Abstract
Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.
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Affiliation(s)
- Florence Wianny
- Inserm, U846, Stem Cell and Brain Research Institute, 18 Avenue Doyen Lépine, 69500 Bron, France.
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21
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Red blood cell production from immortalized progenitor cell line. Int J Hematol 2010; 93:5-9. [PMID: 21184289 DOI: 10.1007/s12185-010-0742-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2010] [Revised: 11/24/2010] [Accepted: 11/30/2010] [Indexed: 10/18/2022]
Abstract
The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and have established many immortalized erythroid progenitor cell lines so far. Although their precise characteristics varied among cell lines, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. Considering the number of human ES cell lines that have been established so far and the number of induced pluripotent stem cell lines that will be established in future, the intensive testing of a number of these lines for establishing immortalized erythroid progenitor cell lines may allow the establishment of such cell lines similar to the mouse erythroid progenitor cell lines.
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22
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Fridley KM, Fernandez I, Li MTA, Kettlewell RB, Roy K. Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors. Tissue Eng Part A 2010; 16:3285-98. [PMID: 20528675 DOI: 10.1089/ten.tea.2010.0166] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.
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Affiliation(s)
- Krista M Fridley
- Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA
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23
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Abstract
Recent studies of the effects of developmental iron deficiency (ID) and iron deficiency anemia in nonhuman primates have provided new insights into this widespread and well-recognized human nutritional deficiency. The rhesus monkey was the animal model in these experiments, which used extensive hematological and behavioral evaluations in addition to noninvasive brain measures. Two important findings were as follows: 1) different behavioral consequences depending on the timing of ID relative to brain developmental stages and 2) the potential for long-lasting changes in brain iron regulatory systems. Further work in this model, including integration with studies in humans and in laboratory rodents, is ongoing.
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Affiliation(s)
- Mari S Golub
- Brain Mind and Behavior Unit, California National Primate Research Center, University of California, Davis, CA, USA.
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Sakamoto H, Tsuji-Tamura K, Ogawa M. Hematopoiesis from pluripotent stem cell lines. Int J Hematol 2010; 91:384-91. [PMID: 20169427 DOI: 10.1007/s12185-010-0519-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2009] [Accepted: 11/12/2009] [Indexed: 01/20/2023]
Abstract
Embryonic stem cells (ESCs) can differentiate into various types of hematopoietic cells (HPCs) when placed in an appropriate environment. Various methods for the differentiation of ESCs into specific HPC lineages have been developed using mouse ESCs. These ESC-differentiation methods have been utilized also as an in vitro model to investigate hematopoiesis in embryos and they provided critical perceptions into it. These methods have been adapted for use with human ESCs, which have the possibility of being employed in regenerative medicine; further improvement of these methods may lead to the efficient production of HPCs for use in transfusions. The generation of transplantable hematopoietic stem cells is a medical goal that is still difficult to achieve. Recently, induced pluripotent stem (iPS) cells have been established from differentiated cells. Thereby, iPS cells have expanded further possibilities of the use of pluripotent stem cell lines in clinical application. Indeed, iPS cells have been established from cells with disease genes and those which have undergone reprogramming and targeting have generated phenotypically normal HPCs. Here, we mainly summarize the recent progress in research on hematopoiesis conducted with ESCs and iPS cells.
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Affiliation(s)
- Hiroshi Sakamoto
- Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
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25
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Maeda T, Ito K, Merghoub T, Poliseno L, Hobbs RM, Wang G, Dong L, Maeda M, Dore LC, Zelent A, Luzzatto L, Teruya-Feldstein J, Weiss MJ, Pandolfi PP. LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis. Dev Cell 2009; 17:527-40. [PMID: 19853566 DOI: 10.1016/j.devcel.2009.09.005] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2009] [Revised: 06/11/2009] [Accepted: 09/18/2009] [Indexed: 12/15/2022]
Abstract
GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, our data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF.
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Affiliation(s)
- Takahiro Maeda
- Cancer Biology and Genetics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
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26
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Yoshimoto M, Heike T, Chang H, Kanatsu-Shinohara M, Baba S, Varnau JT, Shinohara T, Yoder MC, Nakahata T. Bone marrow engraftment but limited expansion of hematopoietic cells from multipotent germline stem cells derived from neonatal mouse testis. Exp Hematol 2009; 37:1400-10. [PMID: 19782120 DOI: 10.1016/j.exphem.2009.09.006] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2009] [Revised: 09/01/2009] [Accepted: 09/21/2009] [Indexed: 12/29/2022]
Abstract
OBJECTIVE Multipotent germline stem (mGS) cells derived from neonatal mouse testis, similar to embryonic stem (ES) cells, differentiate into various types of somatic cells in vitro and produce teratomas after inoculation into mice. In the present work, we examined mGS cells for hematopoietic progenitor potential in vitro and in vivo. MATERIALS AND METHODS mGS cells were differentiated on OP9 stromal cells and induced into Flk1(+) cells. Flk1(+) cells were sorted and replated on OP9 stromal cells with various cytokines and emerging hematopoietic cells were analyzed for lineage marker expression by fluorescein-activated cell sorting, progenitor activity by colony assay, and stem cell transplantation assay. RESULTS mGS cells, like ES cells, produce hematopoietic progenitors, including both primitive and definitive erythromyeloid, megakaryocyte, and B- and T-cell lineages via Flk1(+) progenitors. When transplanted into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficient (NOD/SCID) gammac(null) mice directly, mGS-derived green fluorescent protein (GFP)-positive cells were detected 4 months later in the BM and spleen. GFP(+) donor cells were also identified in the Hoechst33342 side population, a feature of hematopoietic stem cells. However, these mGS-derived hematopoietic cells did not proliferate in vivo, even after exposure to hematopoietic stressors, such as 5-fluorouracil (5FU) injection or serial transplantation. CONCLUSION mGS cells produced multipotent hematopoietic progenitor cells with myeloid and lymphoid lineage potential in vitro and localized in the BM after intra-BM injection but, like ES cells, failed to expand or show stem cell repopulating ability in vivo.
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28
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Niwa A, Umeda K, Chang H, Saito M, Okita K, Takahashi K, Nakagawa M, Yamanaka S, Nakahata T, Heike T. Orderly hematopoietic development of induced pluripotent stem cells via Flk-1(+) hemoangiogenic progenitors. J Cell Physiol 2009; 221:367-77. [PMID: 19562687 DOI: 10.1002/jcp.21864] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics, are generated by the introduction of combinations of specific transcription factors. Little is known about the differentiation of iPS cells in vitro. Here we demonstrate that murine iPS cells produce various hematopoietic cell lineages when incubated on a layer of OP9 stromal cells. During this differentiation, iPS cells went through an intermediate stage consisting of progenitor cells that were positive for the early mesodermal marker Flk-1 and for the sequential expression of other genes that are associated with hematopoietic and endothelial development. Flk-1(+) cells differentiated into primitive and definitive hematopoietic cells, as well as into endothelial cells. Furthermore, Flk-1(+) populations contained common bilineage progenitors that could generate both hematopoietic and endothelial lineages from single cells. Our results demonstrate that iPS cell-derived cells, like ES cells, can follow a similar hematopoietic route to that seen in normal embryogenesis. This finding highlights the potential use of iPS cells in clinical areas such as regenerative medicine, disease investigation, and drug screening.
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Affiliation(s)
- Akira Niwa
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Priddle H, Jones DRE, Burridge PW, Patient R. Hematopoiesis from Human Embryonic Stem Cells: Overcoming the Immune Barrier in Stem Cell Therapies. Stem Cells 2009; 24:815-24. [PMID: 16306149 DOI: 10.1634/stemcells.2005-0356] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.
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Affiliation(s)
- Helen Priddle
- Department of Obstetrics and Gynaecology, School of Human Development, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom.
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Navara CS, Redinger C, Mich-Basso J, Oliver S, Ben-Yehudah A, Castro C, Simerly C. Derivation and characterization of nonhuman primate embryonic stem cells. ACTA ACUST UNITED AC 2008; Chapter 1:Unit 1A.1. [PMID: 18785157 DOI: 10.1002/9780470151808.sc01a01s1] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Embryonic stem (ES) cells are a powerful research tool enabling the generation of mice with custom genetics, the study of the earliest stages of mammalian differentiation in vitro and, with the isolation of human ES cells, the potential of cell-based therapies for a number of diseases including Parkinson's and Type 1 diabetes. ES cells isolated from nonhuman primates (nhpES cells) offer the opportunity to ethically test the developmental potential of primate ES cells in chimeric offspring. If these cells have similar potency to mouse ES cells, this may open a new era of primate models of human disease. Nonhuman primates are the perfect model system for the preclinical testing of ES cell-derived therapies. In this unit, we describe methods for the derivation and characterization of nonhuman primate ES cells. With these protocols, the investigator will be able to isolate nhpES cells and perform the necessary tests to confirm the pluripotent phenotype.
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Nakahara M, Matsuyama S, Saeki K, Nakamura N, Saeki K, Yogiashi Y, Yoneda A, Koyanagi M, Kondo Y, Yuo A. A Feeder-Free Hematopoietic Differentiation System with Generation of Functional Neutrophils from Feeder- and Cytokine-Free Primate Embryonic Stem Cells. CLONING AND STEM CELLS 2008; 10:341-54. [DOI: 10.1089/clo.2007.0068] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Masako Nakahara
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Satoko Matsuyama
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Kumiko Saeki
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Naoko Nakamura
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Koichi Saeki
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Yoshiko Yogiashi
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Asako Yoneda
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Makoto Koyanagi
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
| | - Yasushi Kondo
- Regenerative Medicine Group, Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan
| | - Akira Yuo
- Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan
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Kishi Y, Tanaka Y, Shibata H, Nakamura S, Takeuchi K, Masuda S, Ikeda T, Muramatsu SI, Hanazono Y. Variation in the Incidence of Teratomas after the Transplantation of Nonhuman Primate ES Cells into Immunodeficient Mice. Cell Transplant 2008; 17:1095-1102. [DOI: 10.3727/096368908786991560] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/?cnull (NOG) mice, teratomas developed in all the animals transplanted with 1 × 105 or more cells, but were not observed in any mouse transplanted with 1 × 103 cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 × 105. When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 × 105. When cynomolgus ES cell-derived progenitor cells (1 × 106), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.
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Affiliation(s)
- Yukiko Kishi
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
| | - Yujiro Tanaka
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
| | - Hiroaki Shibata
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
- Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki 305-0843, Japan
| | - Shinichiro Nakamura
- The Corporation for Production and Research of Laboratory Primates, Ibaraki 300-2658, Japan
| | - Koichi Takeuchi
- Department of Anatomy, Jichi Medical University, Tochigi 329-0498, Japan
| | - Shigeo Masuda
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
| | - Tamako Ikeda
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
| | - Shin-Ichi Muramatsu
- Division of Neurology, Department of Internal Medicine, Jichi Medical University, Tochigi 329-0498, Japan
| | - Yutaka Hanazono
- Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
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Ji J, Vijayaragavan K, Bosse M, Menendez P, Weisel K, Bhatia M. OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells. Stem Cells 2008; 26:2485-95. [PMID: 18669904 DOI: 10.1634/stemcells.2008-0642] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45(+)CD34(+) cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45(+)CD34(-)) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- Junfeng Ji
- Faculty of Health Sciences, Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, Canada
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Ema H, Nakauchi H. Bloodlines of haematopoietic stem cell research in Japan. Philos Trans R Soc Lond B Biol Sci 2008; 363:2089-97. [PMID: 18375375 DOI: 10.1098/rstb.2008.2263] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Haematopoietic stem cells (HSCs) can supply all blood cells throughout the adult life of individuals. Based on this property, HSCs have been used for bone marrow and cord blood transplantation. Among various stem cells, HSCs were recognized earliest and were studied most extensively, providing a model for other stem cells. Knowledge of HSC regulation has rapidly accumulated of late. Contributions of scientists in Japan to progress HSC biology are here briefly overviewed. Focusing on the original work accomplished in Japan in the last two decades, people who have led such activities are introduced and their relationships with one another are sketched.
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Affiliation(s)
- Hideo Ema
- Laboratory of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Hiroyama T, Miharada K, Sudo K, Danjo I, Aoki N, Nakamura Y. Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells. PLoS One 2008; 3:e1544. [PMID: 18253492 PMCID: PMC2212133 DOI: 10.1371/journal.pone.0001544] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2007] [Accepted: 01/03/2008] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. METHODOLOGY/PRINCIPAL FINDINGS We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. CONCLUSION/SIGNIFICANCE To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.
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Affiliation(s)
- Takashi Hiroyama
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
| | - Kenichi Miharada
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
| | - Kazuhiro Sudo
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
| | - Inaho Danjo
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
| | - Naoko Aoki
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
- * To whom correspondence should be addressed. E-mail:
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36
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Nakamura Y. In vitro Production of Transfusable Red Blood Cells. Biotechnol Genet Eng Rev 2008; 25:187-201. [DOI: 10.5661/bger-25-187] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood 2007; 111:2400-8. [PMID: 18024790 DOI: 10.1182/blood-2007-07-102087] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.
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Ma F, Kambe N, Wang D, Shinoda G, Fujino H, Umeda K, Fujisawa A, Ma L, Suemori H, Nakatsuji N, Miyachi Y, Torii R, Tsuji K, Heike T, Nakahata T. Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells. Stem Cells 2007; 26:706-14. [PMID: 17991916 DOI: 10.1634/stemcells.2007-0348] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Conditions that influence the selective development or recruitment of connective tissue-type and mucosal-type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta-gonad-mesonephros-derived stromal cell line AGM-S1 differentiated into cobblestone (CS)-like cells by day 10-15. When replated onto fresh AGM-S1 with the addition of stem cell factor, interleukin-6, and Flt3 ligand, these CS-like cells displayed robust growth and generated almost 100% tryptase/chymase double-positive MCs within 3 weeks. At all time points, the percentage of tryptase-positive cells did not exceed that of chymase-positive cells. These ES-derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA-DR- and coexpressed a high-affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES-derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES-derived MCs have the phenotype of functionally mature connective tissue-type MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue-type MCs, which may be independent from the developmental pathway of mucosal-type MCs.
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Affiliation(s)
- Feng Ma
- Department of Pediatrics, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Rajesh D, Chinnasamy N, Mitalipov SM, Wolf DP, Slukvin I, Thomson JA, Shaaban AF. Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells. Stem Cells 2007; 25:490-9. [PMID: 17284653 DOI: 10.1634/stemcells.2006-0277] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.
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Affiliation(s)
- Deepika Rajesh
- Department of Surgery, University of Wisconsin Medical School, K4/760 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792-7375, USA
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Baba S, Heike T, Umeda K, Iwasa T, Kaichi S, Hiraumi Y, Doi H, Yoshimoto M, Kanatsu-Shinohara M, Shinohara T, Nakahata T. Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells. Stem Cells 2007; 25:1375-83. [PMID: 17322104 DOI: 10.1634/stemcells.2006-0574] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- Shiro Baba
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Tsuchiya A, Heike T, Baba S, Fujino H, Umeda K, Matsuda Y, Nomoto M, Ichida T, Aoyagi Y, Nakahata T. Long-term culture of postnatal mouse hepatic stem/progenitor cells and their relative developmental hierarchy. Stem Cells 2007; 25:895-902. [PMID: 17218396 DOI: 10.1634/stemcells.2006-0558] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature alpha-fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1+ cells. As mice aged, the frequency of SP and sca-1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%-40% of the SP cells expressed sca-1, but only a few sca-1+ cells were also SP cells. Analysis of colonies derived from single SP or sca-1+ cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1+ cells, whereas sca-1+ cells generated only sca-1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca-1+ cells. In regenerating livers, ABCG2+ cells and sca-1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.
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Affiliation(s)
- Atsunori Tsuchiya
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Abstract
Embryonic stem cells represent a pluripotent population of cells capable of self-renewal, large-scale expansion, and differentiation in various cell lineages including cells of hematopoietic lineage. In this chapter, we describe a three-step cell culture method for directed differentiation of human embryonic stem cells (hESCs) to dendritic cells (DCs) that includes (1) hESC differentiation into hematopoietic progenitors by coculture with OP9 stromal cells, (2) expansion of myeloid DC precursors in suspension bulk cultures with granulocyte monocyte-colony stimulating factor (GM-CSF), and (3) differentiation of myeloid precursors to DCs in the serum-free medium with GM-CSF and interleukin-4 (IL-4). The method employs cell culture conditions selecting an almost pure population of myeloid DC precursors and does not require isolation of hematopoietic progenitors. With this method, hESCs can be differentiated to functional DCs within 30 days at an efficiency of at least four DCs per single undifferentiated hESC. Directed differentiation of DCs from hESCs could be useful for studying cellular and molecular mechanisms of DC development and potentially for the generation of antigen-presenting cells for cellular immunotherapy.
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Trivedi P, Hematti P. Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells. Exp Hematol 2007; 35:146-54. [PMID: 17198883 DOI: 10.1016/j.exphem.2006.09.003] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2006] [Revised: 09/06/2006] [Accepted: 09/07/2006] [Indexed: 12/24/2022]
Abstract
OBJECTIVE Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. MATERIALS AND METHODS We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. RESULTS We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+) hematopoietic and CD73(+) MSC generation follows a similar pattern. We sorted the CD34(+) cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. CONCLUSIONS OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.
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Affiliation(s)
- Parul Trivedi
- Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA
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Saito K, Yoshikawa M, Ouji Y, Moriya K, Nishiofuku M, Ueda S, Hayashi N, Ishizaka S, Fukui H. Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells. World J Gastroenterol 2006; 12:6818-27. [PMID: 17106931 PMCID: PMC4087437 DOI: 10.3748/wjg.v12.i42.6818] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes.
METHODS: Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone.
RESULTS: The mRNA expression of α-fetoprotein, albumin (ALB), α-1-antitrypsin, and hepatocyte nuclear factor 4α was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated.
CONCLUSION: MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.
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Affiliation(s)
- Ko Saito
- Department of Gastroenterology and Hepatology, Nara Medical University, Nara, Japan
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Shinoda G, Umeda K, Heike T, Arai M, Niwa A, Ma F, Suemori H, Luo HY, Chui DHK, Torii R, Shibuya M, Nakatsuji N, Nakahata T. alpha4-Integrin(+) endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells. Blood 2006; 109:2406-15. [PMID: 17090646 DOI: 10.1182/blood-2006-06-031039] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. In this study, we demonstrated that VE-cadherin(+)CD45(-) endothelial cells derived from nonhuman primate embryonic stem cells are able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. Single bipotential progenitors for hematopoietic and endothelial lineages are included in this endothelial cell population. Furthermore, hemogenic activity of these endothelial cells is observed exclusively in the alpha4-integrin(+) subpopulation; bipotential progenitors are 4-fold enriched in this subpopulation. The kinetics of this hemogenic subpopulation is similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo in that they emerge for only a limited time. We suggest that VE-cadherin(+)CD45(-)alpha4-integrin(+) endothelial cells are involved in primitive and definitive hematopoiesis during primate embryogenesis, though VE-cadherin(-)CD45(-)alpha4-integrin(+) cells are the primary sources for primitive hematopoiesis.
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Affiliation(s)
- Gen Shinoda
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Japan
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Shaaban AF, Kim HB, Gaur L, Liechty KW, Flake AW. Prenatal transplantation of cytokine-stimulated marrow improves early chimerism in a resistant strain combination but results in poor long-term engraftment. Exp Hematol 2006; 34:1278-87. [PMID: 16939821 PMCID: PMC3096442 DOI: 10.1016/j.exphem.2006.05.007] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2006] [Revised: 05/08/2006] [Accepted: 05/08/2006] [Indexed: 01/17/2023]
Abstract
OBJECTIVE In the absence of immunodeficiency, only microchimerism (<0.1%) has been achieved in human fetal recipients or nonhuman primates following in utero hematopoietic cell transplantation (IUHCT). We hypothesized that enhanced long-term engraftment might be more reliably achieved in microchimeric systems if higher levels of chimerism existed during development of adaptive immunity. To evaluate this hypothesis, we stimulated the donor cells with vascular endothelial growth factor (VEGF) and stem cell factor (SCF) prior to IUHCT in a chimerism-resistant murine strain combination. METHODS Donor Balb/c marrow was cultured in media with or without VEGF and SCF supplementation for 12 hours prior to IUHCT into B6 fetuses at 14 days postcoitum (dpc). Donor cell phenotype, homing, and chimerism were assessed at short and long-term time points and transplanted animals received skin allografts at 8 weeks. RESULTS In pretreated allogeneic recipients, early chimerism rates were more than double that of controls (71% vs 33%, p = 0.01). These differences were associated with higher numbers of pretransplant donor cell colony-forming cells without change in donor cell homing. Despite prolonged skin allograft survival for pretreated recipients compared with controls (mean survival = 20.8 vs 8.2 days, p < 0.001), long-term engraftment was unchanged. CONCLUSIONS These findings demonstrate that higher levels of early chimerism in recipients of cytokine-stimulated marrow result in improved short-term chimerism and tolerance. Future studies are needed to confirm the existence of a "threshold" level of chimerism necessary to sustain long-term engraftment.
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Affiliation(s)
- Aimen F Shaaban
- Laboratory for Fetal Cellular Therapy, Department of Surgery, University of Wisconsin Medical School, Madison, WI 53792-7375, USA.
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Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, Imoto H, Izawa K, Dong Y, Hashiguchi T, Soda Y, Maeda T, Suehiro Y, Tanioka Y, Nakazaki Y, Tani K. Tal1/Scl Gene Transduction Using a Lentiviral Vector Stimulates Highly Efficient Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells. Stem Cells 2006; 24:2014-22. [PMID: 16728561 DOI: 10.1634/stemcells.2005-0499] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.
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Affiliation(s)
- Ryo Kurita
- Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Fukuoka 812-8582, Japan
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Nakatsuji N. Establishment and manipulation of monkey and human embryonic stem cell lines for biomedical research. ERNST SCHERING RESEARCH FOUNDATION WORKSHOP 2006:15-26. [PMID: 16080284 DOI: 10.1007/3-540-37644-5_2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
We have established several embryonic stem (ES) cell lines of the cynomolgus monkey. They maintain the normal karyotype and pluripotency in culture for long periods. We obtained government approval and grants to produce human ES cell lines from frozen surplus embryos in April 2002. We have established and characterized three human ES cell lines (KhES-1, KhES-2, KhES-3). We started distribution of human ES cells to other research groups in March 2004. It would be important to produce genetically modified monkey and human ES cells for various purposes. After improvement of the transfection and selection methods, we have produced monkey ES cells with integrated transgenes at efficient and reliable rates. We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES cells. Such reprogramming could be used to produce pluripotent stem cells for each patient without therapeutic cloning, which would raise ethical concerns.
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Affiliation(s)
- N Nakatsuji
- Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Japan.
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Umeda K, Heike T, Nakata-Hizume M, Niwa A, Arai M, Shinoda G, Ma F, Suemori H, Luo HY, Chui DHK, Torii R, Shibuya M, Nakatsuji N, Nakahata T. Sequential analysis of alpha- and beta-globin gene expression during erythropoietic differentiation from primate embryonic stem cells. Stem Cells 2006; 24:2627-36. [PMID: 16888280 DOI: 10.1634/stemcells.2006-0199] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.
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Affiliation(s)
- Katsutsugu Umeda
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Srivastava AS, Kaushal S, Mishra R, Lane TA, Carrier E. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro. Biochem Biophys Res Commun 2006; 346:508-16. [PMID: 16764825 DOI: 10.1016/j.bbrc.2006.05.130] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2006] [Accepted: 05/22/2006] [Indexed: 11/22/2022]
Abstract
Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.
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Affiliation(s)
- Anand S Srivastava
- Department of Medicine, Moores UCSD Cancer Center, University of California San Diego, 92093-0820, USA
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