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1
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Furniss JA, Tarassova N, Poole AW. Platelet generation in vivo and in vitro. Blood 2024; 144:2283-2294. [PMID: 39357055 DOI: 10.1182/blood.2024024601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2024] [Revised: 08/08/2024] [Accepted: 09/20/2024] [Indexed: 10/04/2024] Open
Abstract
ABSTRACT Platelets play crucial roles in hemostasis, thrombosis, and immunity, but our understanding of their complex biogenesis (thrombopoiesis) is currently incomplete. Deeper insight into the mechanisms of platelet biogenesis inside and outside the body is fundamental for managing hematological disorders and for the development of novel cell-based therapies. In this article, we address the current understanding of in vivo thrombopoiesis, including mechanisms of platelet generation from megakaryocytes (proplatelet formation, cytoplasmic fragmentation, and membrane budding) and their physiological location. Progress has been made in replicating these processes in vitro for potential therapeutic application, notably in platelet transfusion and bioengineering of platelets for novel targeted therapies. The current platelet-generating systems and their limitations, particularly yield, scalability, and functionality, are discussed. Finally, we highlight the current controversies and challenges in the field that need to be addressed to achieve a full understanding of these processes, in vivo and in vitro.
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Affiliation(s)
- Jonathan A Furniss
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Nathalie Tarassova
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Alastair W Poole
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
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2
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Kweon S, Kim S, Choi HS, Jo K, Park JM, Baek EJ. Current status of platelet manufacturing in 3D or bioreactors. Biotechnol Prog 2023; 39:e3364. [PMID: 37294031 DOI: 10.1002/btpr.3364] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Revised: 05/09/2023] [Accepted: 05/09/2023] [Indexed: 06/10/2023]
Abstract
Blood shortages for transfusion are global issues of grave concern. As in vitro manufactured platelets are promising substitutes for blood donation, recent research has shown progresses including different cell sources, different bioreactors, and three-dimensional materials. The first-in-human clinical trial of cultured platelets using induced pluripotent stem cell-derived platelets began in Japan and demonstrated its quality, safety, and efficacy. A novel bioreactor with fluid motion for platelet production has been reported. Herein, we discuss various cell sources for blood cell production, recent advances in manufacturing processes, and clinical applications of cultured blood.
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Affiliation(s)
- Soonho Kweon
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
| | - Suyeon Kim
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
| | - Hye Sook Choi
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
| | - Kyeongwon Jo
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
| | - Ju Mi Park
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
| | - Eun Jung Baek
- Department of Research and Development, ArtBlood Inc, Seoul, Republic of Korea
- Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
- Department of Laboratory Medicine, Hanyang University College of Medicine, Seoul, Republic of Korea
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3
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Zhu M, Wang Q, Gu T, Han Y, Zeng X, Li J, Dong J, Huang H, Qian P. Hydrogel-based microenvironment engineering of haematopoietic stem cells. Cell Mol Life Sci 2023; 80:49. [PMID: 36690903 PMCID: PMC11073069 DOI: 10.1007/s00018-023-04696-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 11/06/2022] [Accepted: 01/08/2023] [Indexed: 01/25/2023]
Abstract
Haematopoietic Stem cells (HSCs) have the potential for self-renewal and multilineage differentiation, and their behaviours are finely tuned by the microenvironment. HSC transplantation (HSCT) is widely used in the treatment of haematologic malignancies while limited by the quantity of available HSCs. With the development of tissue engineering, hydrogels have been deployed to mimic the HSC microenvironment in vitro. Engineered hydrogels influence HSC behaviour by regulating mechanical strength, extracellular matrix microstructure, cellular ligands and cytokines, cell-cell interaction, and oxygen concentration, which ultimately facilitate the acquisition of sufficient HSCs. Here, we review recent advances in the application of hydrogel-based microenvironment engineering of HSCs, and provide future perspectives on challenges in basic research and clinical practice.
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Affiliation(s)
- Meng Zhu
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Qiwei Wang
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Tianning Gu
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
- Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yingli Han
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Xin Zeng
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Jinxin Li
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Jian Dong
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - He Huang
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.
- Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
| | - Pengxu Qian
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.
- Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.
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4
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Chen SJ, Sugimoto N, Eto K. Ex vivo manufacturing of platelets: beyond the first-in-human clinical trial using autologous iPSC-platelets. Int J Hematol 2023; 117:349-355. [PMID: 36574167 PMCID: PMC9792917 DOI: 10.1007/s12185-022-03512-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2022] [Revised: 12/05/2022] [Accepted: 12/06/2022] [Indexed: 12/28/2022]
Abstract
Platelet transfusion is a common clinical approach to providing platelets to patients suffering from thrombocytopenia or other ailments that require an additional platelet source. However, a stable supply of platelet products is challenged by aging societies, pandemics, and other factors. Many groups have made extensive efforts toward the in vitro generation of platelets for clinical application. We established immortalized megakaryocyte progenitor cell lines (imMKCLs) from human induced pluripotent stem cells (iPSCs) and achieved clinical-scale manufacturing of iPSC-derived platelets (iPSC-PLTs) from them by identifying turbulent flow as a key physical condition. We later completed the iPLAT1 study, the first-in-human clinical trial using autologous iPSC-PLTs. This review summarizes current findings on the ex vivo generation of iPSC-PLTs that led to the iPLAT1 study and beyond. We also discuss new insights regarding the heterogeneity of megakaryocytes and the implications for the ex vivo generation of iPSC-PLTs.
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Affiliation(s)
- Si Jing Chen
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
| | - Naoshi Sugimoto
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Koji Eto
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan. .,Department of Regenerative Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
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5
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Tang A, Mendelson A. Recent lessons learned for ex-vivo platelet production. Curr Opin Hematol 2021; 28:424-430. [PMID: 34232141 PMCID: PMC8490274 DOI: 10.1097/moh.0000000000000662] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PURPOSE OF REVIEW Platelet transfusion can be life-saving but carries a risk of infection or alloimmunization and is limited by insufficient donor sources and restricted unit shelf life. Generating sufficient platelets in vitro to replace a unit of collected blood remains a challenge. Here, we examine the latest advances in the regulation of megakaryocyte maturation and expansion along with platelet formation and survival. We also discuss alternative therapies investigated to induce platelet production. RECENT FINDINGS Recent studies examined candidate niche cells in the bone marrow microenvironment for promoting platelet formation and developed an explant-based bioreactor to enhance platelet production ex vivo. Chemical inhibitors were examined for their ability to promote megakaryocyte maturation and expansion. Microparticles from megakaryocytes or platelets were found to improve megakaryocyte maturation and platelet formation. Membrane budding was identified as a novel mode of platelet formation. Lastly, a chemical inhibitor to improve cold-stored platelets was identified. SUMMARY Recent advances in the regulation of megakaryocyte expansion and platelet production provide exciting promise for the development of improved approaches to generate platelets in vitro. These findings bring the field one step closer to achieving the ultimate goal of creating a unit of platelets without the need for donation.
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Affiliation(s)
- Alice Tang
- Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY
| | - Avital Mendelson
- Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY
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6
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Di Buduo CA, Aguilar A, Soprano PM, Bocconi A, Miguel CP, Mantica G, Balduini A. Latest culture techniques: cracking the secrets of bone marrow to mass-produce erythrocytes and platelets ex vivo. Haematologica 2021; 106:947-957. [PMID: 33472355 PMCID: PMC8017859 DOI: 10.3324/haematol.2020.262485] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Indexed: 12/13/2022] Open
Abstract
Since the dawn of medicine, scientists have carefully observed, modeled and interpreted the human body to improve healthcare. At the beginning there were drawings and paintings, now there is three-dimensional modeling. Moving from two-dimensional cultures and towards complex and relevant biomaterials, tissue-engineering approaches have been developed in order to create three-dimensional functional mimics of native organs. The bone marrow represents a challenging organ to reproduce because of its structure and composition that confer it unique biochemical and mechanical features to control hematopoiesis. Reproducing the human bone marrow niche is instrumental to answer the growing demand for human erythrocytes and platelets for fundamental studies and clinical applications in transfusion medicine. In this review, we discuss the latest culture techniques and technological approaches to obtain functional platelets and erythrocytes ex vivo. This is a rapidly evolving field that will define the future of targeted therapies for thrombocytopenia and anemia, but also a long-term promise for new approaches to the understanding and cure of hematologic diseases.
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Affiliation(s)
| | - Alicia Aguilar
- Department of Molecular Medicine, University of Pavia, Pavia
| | - Paolo M Soprano
- Department of Molecular Medicine, University of Pavia, Pavia
| | - Alberto Bocconi
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Department of Chemistry, Materials and Chemical Engineering G. Natta, Politecnico di Milano, Milano
| | | | | | - Alessandra Balduini
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Department of Biomedical Engineering, Tufts University, Medford, MA
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7
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Generation and manipulation of human iPSC-derived platelets. Cell Mol Life Sci 2021; 78:3385-3401. [PMID: 33439272 PMCID: PMC7804213 DOI: 10.1007/s00018-020-03749-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 12/01/2020] [Accepted: 12/23/2020] [Indexed: 12/17/2022]
Abstract
The discovery of iPSCs has led to the ex vivo production of differentiated cells for regenerative medicine. In the case of transfusion products, the derivation of platelets from iPSCs is expected to complement our current blood-donor supplied transfusion system through donor-independent production with complete pathogen-free assurance. This derivation can also overcome alloimmune platelet transfusion refractoriness by resulting in autologous, HLA-homologous or HLA-deficient products. Several developments were necessary to produce a massive number of platelets required for a single transfusion. First, expandable megakaryocytes were established from iPSCs through transgene expression. Second, a turbulent-type bioreactor with improved platelet yield and quality was developed. Third, novel drugs that enabled efficient feeder cell-free conditions were developed. Fourth, the platelet-containing suspension was purified and resuspended in an appropriate buffer. Finally, the platelet product needed to be assured for competency and safety including non-tumorigenicity through in vitro and in vivo preclinical tests. Based on these advancements, a clinical trial has started. The generation of human iPSC-derived platelets could evolve transfusion medicine to the next stage and assure a ubiquitous, safe supply of platelet products. Further, considering the feasibility of gene manipulations in iPSCs, other platelet products may bring forth novel therapeutic measures.
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8
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Ghanbari OB, Soleimani M, Shahidi M, Ghiass MA, Enderami SE, Dorgalaleh A. Differentiation of human induced pluripotent stem cells to megakaryocyte lineage by using 3D bioreactor, microfluidic system and acellular rat lung. Biochem Eng J 2021. [DOI: 10.1016/j.bej.2020.107822] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
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9
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Martínez-Botía P, Acebes-Huerta A, Seghatchian J, Gutiérrez L. On the Quest for In Vitro Platelet Production by Re-Tailoring the Concepts of Megakaryocyte Differentiation. ACTA ACUST UNITED AC 2020; 56:medicina56120671. [PMID: 33287459 PMCID: PMC7761839 DOI: 10.3390/medicina56120671] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 11/26/2020] [Accepted: 11/30/2020] [Indexed: 12/14/2022]
Abstract
The demand of platelet transfusions is steadily growing worldwide, inter-donor variation, donor dependency, or storability/viability being the main contributing factors to the current global, donor-dependent platelet concentrate shortage concern. In vitro platelet production has been proposed as a plausible alternative to cover, at least partially, the increasing demand. However, in practice, such a logical production strategy does not lack complexity, and hence, efforts are focused internationally on developing large scale industrial methods and technologies to provide efficient, viable, and functional platelet production. This would allow obtaining not only sufficient numbers of platelets but also functional ones fit for all clinical purposes and civil scenarios. In this review, we cover the evolution around the in vitro culture and differentiation of megakaryocytes into platelets, the progress made thus far to bring the culture concept from basic research towards good manufacturing practices certified production, and subsequent clinical trial studies. However, little is known about how these in vitro products should be stored or whether any safety measure should be implemented (e.g., pathogen reduction technology), as well as their quality assessment (how to isolate platelets from the rest of the culture cells, debris, microvesicles, or what their molecular and functional profile is). Importantly, we highlight how the scientific community has overcome the old dogmas and how the new perspectives influence the future of platelet-based therapy for transfusion purposes.
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Affiliation(s)
- Patricia Martínez-Botía
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain; (P.M.-B.); (A.A.-H.)
- Department of Medicine, University of Oviedo, 33003 Oviedo, Spain
| | - Andrea Acebes-Huerta
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain; (P.M.-B.); (A.A.-H.)
| | - Jerard Seghatchian
- International Consultancy in Strategic Safety/Quality Improvements of Blood-Derived Bioproducts and Suppliers Quality Audit/Inspection, London NW3 3AA, UK;
| | - Laura Gutiérrez
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain; (P.M.-B.); (A.A.-H.)
- Department of Medicine, University of Oviedo, 33003 Oviedo, Spain
- Correspondence:
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10
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Strassel C, Lanza F, Gachet C. Plaquettes sanguines de culture : état de l’art. BULLETIN DE L'ACADÉMIE NATIONALE DE MÉDECINE 2020; 204:971-980. [PMID: 33078027 PMCID: PMC7556249 DOI: 10.1016/j.banm.2020.10.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 10/03/2020] [Indexed: 11/09/2022]
Abstract
Les plaquettes sanguines sont des éléments anucléés du sang. D’un diamètre de 2 à 3 μm, ce sont les plus petits éléments figurés du sang. Alors que leur rôle principal est d’arrêter ou prévenir les saignements, elles sont également impliquées dans d’autres fonctions, comme l’immunité, l’inflammation ou la progression tumorale. L’essor des biotechnologies et les connaissances acquises sur les mécanismes qui régulent la biogénèse des plaquettes permettent aujourd’hui d’envisager la production de plaquettes de culture. Dès lors, ce type de produit pourrait avoir sa place pour relever un certain nombre de défis transfusionnels comme l’allo-immunisation ou les états réfractaires. Cependant les rendements de culture restent faibles et de nombreux obstacles doivent encore être franchis avant d’envisager une application en transfusion. Cet article recense les arguments qui motivent la production de plaquettes de culture à visée transfusionnelle et récapitule les principales avancées dans le domaine tout en soulignant ses limites.
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11
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Yang J, Luan J, Shen Y, Chen B. Developments in the production of platelets from stem cells (Review). Mol Med Rep 2020; 23:7. [PMID: 33179095 PMCID: PMC7673345 DOI: 10.3892/mmr.2020.11645] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Accepted: 10/13/2020] [Indexed: 01/01/2023] Open
Abstract
Platelets are small pieces of cytoplasm that have become detached from the cytoplasm of mature megakaryocytes (MKs) in the bone marrow. Platelets modulate vascular system integrity and serve important role, particularly in hemostasis. With the rapid development of clinical medicine, the demand for platelet transfusion as a life‑saving intervention increases continuously. Stem cell technology appears to be highly promising for transfusion medicine, and the generation of platelets from stem cells would be of great value in the clinical setting. Furthermore, several studies have been undertaken to investigate the potential of producing platelets from stem cells. Initial success has been achieved in terms of the yields and function of platelets generated from stem cells. However, the requirements of clinical practice remain unmet. The aim of the present review was to focus on several sources of stem cells and factors that induce MK differentiation. Updated information on current research into the genetic regulation of megakaryocytopoiesis and platelet generation was summarized. Additionally, advanced strategies of platelet generation were reviewed and the progress made in this field was discussed.
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Affiliation(s)
- Jie Yang
- Department of Hematology and Oncology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Jianfeng Luan
- Jinling Hospital Department of Blood Transfusion, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China
| | - Yanfei Shen
- Medical School, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Baoan Chen
- Department of Hematology and Oncology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing, Jiangsu 210009, P.R. China
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12
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Harada T, Tsuboi I, Utsunomiya M, Yasuda M, Aizawa S. Kinetics of leukemic cells in 3D culture with stromal cells and with arginine deprivation stress. J Biosci Bioeng 2020; 130:650-658. [PMID: 32861594 DOI: 10.1016/j.jbiosc.2020.07.018] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Revised: 07/08/2020] [Accepted: 07/28/2020] [Indexed: 01/20/2023]
Abstract
Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (-)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562 cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (-). Furthermore, the number of K562 cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (-). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562 cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions in vitro.
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Affiliation(s)
- Tomonori Harada
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610, Japan.
| | - Isao Tsuboi
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610, Japan.
| | - Mizuki Utsunomiya
- Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
| | - Masahiro Yasuda
- Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
| | - Shin Aizawa
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610, Japan.
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13
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Martínez-Botía P, Acebes-Huerta A, Seghatchian J, Gutiérrez L. In vitro platelet production for transfusion purposes: Where are we now? Transfus Apher Sci 2020; 59:102864. [PMID: 32646795 DOI: 10.1016/j.transci.2020.102864] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Over the last decade there has been a worldwide increase in the demand of platelet concentrates (PCs) for transfusion. This is, to a great extent, due to a growing and aging population with the concomitant increase in the incidence of onco-hematological diseases, which require frequent platelet (PLT) transfusions. Currently, PLTs are sourced uniquely from donations, and their storage time is limited only to a few days. The necessity to store PCs at room temperature (to minimize loss of PLT functional integrity), poses a major risk for bacterial contamination. While the implementation of pathogen reduction treatments (PRTs) and new-generation PLT additive solutions have allowed the extension of the shelf life and a safer PLT transfusion product, the concern of PCs shortage still pressures the scientific community to find alternative solutions with the aim of meeting the PLT transfusion increasing demand. In this concise report, we will focus on the efforts made to produce, in in vitro culture, high yields of viable and functional PLTs for transfusion purposes in a cost-effective manner, meeting not only current Good Manufacturing Practices (cGMPs), but also transfusion safety standards.
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Affiliation(s)
- Patricia Martínez-Botía
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain; Dept. of Medicine, University of Oviedo, Spain
| | - Andrea Acebes-Huerta
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain
| | - Jerard Seghatchian
- International Consultancy in Strategic Advices on Safety Improvements of Blood-Derived Bioproducts and Suppliers Quality Audit / Inspection, London, England, UK
| | - Laura Gutiérrez
- Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain; Dept. of Medicine, University of Oviedo, Spain.
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14
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Fujiyama S, Hori N, Sato T, Enosawa S, Murata M, Kobayashi E. Development of an ex vivo xenogeneic bone environment producing human platelet-like cells. PLoS One 2020; 15:e0230507. [PMID: 32255777 PMCID: PMC7138292 DOI: 10.1371/journal.pone.0230507] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 03/03/2020] [Indexed: 12/19/2022] Open
Abstract
The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.
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Affiliation(s)
- Shingo Fujiyama
- Central Research Laboratories, Sysmex Corporation, Kobe-shi, Hyogo, Japan
| | - Nobuyasu Hori
- Central Research Laboratories, Sysmex Corporation, Kobe-shi, Hyogo, Japan
| | - Toshiyuki Sato
- Central Research Laboratories, Sysmex Corporation, Kobe-shi, Hyogo, Japan
| | - Shin Enosawa
- Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan
- Division of Advanced Medical Sciences, National Center for Child Health and Development, Tokyo, Japan
| | - Mitsuru Murata
- Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Eiji Kobayashi
- Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan
- * E-mail:
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15
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Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production. World J Stem Cells 2019; 11:666-676. [PMID: 31616542 PMCID: PMC6789181 DOI: 10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 05/12/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.
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Affiliation(s)
- Xiao-Hua Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yi-Qing Yang
- Faculty of Laboratory Medical Science, Hebei North University, Zhangjiakou 075000, Hebei Province, China
| | - Chi-Yuan Ma
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - En-Kui Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
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16
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Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cellsex vivo: Toward large-scale platelet production. World J Stem Cells 2019. [DOI: dx.doi.org/10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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17
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Ingavle G, Shabrani N, Vaidya A, Kale V. Mimicking megakaryopoiesis in vitro using biomaterials: Recent advances and future opportunities. Acta Biomater 2019; 96:99-110. [PMID: 31319203 DOI: 10.1016/j.actbio.2019.07.025] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2019] [Revised: 07/02/2019] [Accepted: 07/12/2019] [Indexed: 12/24/2022]
Abstract
Presently donor-derived platelets used in the clinic are associated with concerns about adequate availability, expense, risk of bacterial contamination and complications due to immunological reaction. To prevail over our dependence on transfusion of donor-derived platelets, efforts are being made to generate them in vitro. Development of biomaterials that support or mimic bone marrow niche micro-environmental cues could improve the in vitro production of platelets from megakaryocytes (MKs) derived from various stem cell sources. In spite of significant advances in the production of MKs from various stem cell sources using 2D as well as 3D culture approaches in vitro and the development of biomaterials-based platelet systems, yield and quality of these platelets remains unsuitable for clinical use. Thus, in vitro production of clinically useful platelets on a large scale remains an unmet target to date. This review summarizes the most frequently used 2D and 3D approaches to generate MKs and platelets in vitro, emphasizing the importance of mimicking in vivo micro-environment. Further, this review proposes the use of interpenetrating network (IPN) biomaterial-based approach as a promising strategy for improving the generation of MK and platelets in sufficient numbers in vitro. STATEMENT OF SIGNIFICANCE: Thrombocytopenia is one of the major global health and socio-economic problems. Transfusion with donor-derived platelets (PLTs) is the only effective treatment for this condition. However, this approach is limited by factors like short shelf-life of PLTs, PLT activation, alloimmunization, risk of bacterial contamination, infection etc. In vitro generated MKs and PLTs derived from non-donor-dependent sources may help to overcome the platelet transfusion concerns. Here we have reviewed various 2D and 3D strategies used for in vitro generation of MKs and PLTs, with special emphasis on various biomaterial platforms and different physico/chemical cues being used for the purpose. We have also proposed a biomaterial-based approach of using interpenetrating network (IPN) for generating clinically relevant numbers of MKs and PLTs.
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18
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Noetzli LJ, French SL, Machlus KR. New Insights Into the Differentiation of Megakaryocytes From Hematopoietic Progenitors. Arterioscler Thromb Vasc Biol 2019; 39:1288-1300. [PMID: 31043076 PMCID: PMC6594866 DOI: 10.1161/atvbaha.119.312129] [Citation(s) in RCA: 182] [Impact Index Per Article: 30.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Accepted: 04/22/2019] [Indexed: 02/07/2023]
Abstract
Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells, through a hierarchical series of progenitor cells, ultimately to a mature megakaryocyte. Once mature, the megakaryocyte then undergoes a terminal maturation process involving multiple rounds of endomitosis and cytoplasmic restructuring to allow platelet formation. However, recent studies have begun to redefine this hierarchy and shed new light on alternative routes by which hematopoietic stem cells are differentiated into megakaryocytes. In particular, the origin of megakaryocytes, including the existence and hierarchy of megakaryocyte progenitors, has been redefined, as new studies are suggesting that hematopoietic stem cells originate as megakaryocyte-primed and can bypass traditional lineage checkpoints. Overall, it is becoming evident that megakaryopoiesis does not only occur as a stepwise process, but is dynamic and adaptive to biological needs. In this review, we will reexamine the canonical dogmas of megakaryopoiesis and provide an updated framework for interpreting the roles of traditional pathways in the context of new megakaryocyte biology. Visual Overview- An online visual overview is available for this article.
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Affiliation(s)
- Leila J Noetzli
- Division of Hematology, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA
| | - Shauna L French
- Division of Hematology, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA
| | - Kellie R Machlus
- Division of Hematology, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA
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19
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20
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21
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Coller BS. Foreword: A Brief History of Ideas About Platelets in Health and Disease. Platelets 2019. [DOI: 10.1016/b978-0-12-813456-6.09988-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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22
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Strassel C, Gachet C, Lanza F. On the Way to in vitro Platelet Production. Front Med (Lausanne) 2018; 5:239. [PMID: 30211166 PMCID: PMC6120994 DOI: 10.3389/fmed.2018.00239] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Accepted: 08/06/2018] [Indexed: 12/16/2022] Open
Abstract
The severely decreased platelet counts (10–30. 103 platelets/μL) frequently observed in patients undergoing chemotherapy, radiation treatment, or organ transplantation are associated with life-threatening increased bleeding risks. To circumvent these risks, platelet transfusion remains the treatment of choice, despite some limitations which include a limited shelf-life, storage-related deterioration, the development of alloantibodies in recipients and the transmission of infectious diseases. A sustained demand has evolved in recent years for controlled blood products, free of infectious, inflammatory, and immune risks. As a consequence, the challenge for blood centers in the near future will be to ensure an adequate supply of blood platelets, which calls for a reassessment of our transfusion models. To meet this challenge, many laboratories are now turning their research efforts toward the in vitro and customized production of blood platelets. In recent years, there has been a major enthusiasm for the cultured platelet production, as illustrated by the number of reviews that have appeared in recent years. The focus of the present review is to critically asses the arguments put forward in support of the culture of platelets for transfusion purposes. In light of this, we will recapitulate the main advances in this quickly evolving field, while noting the technical limitations to overcome to make cultured platelet a transfusional alternative.
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Affiliation(s)
- Catherine Strassel
- Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Strasbourg, France
| | - Christian Gachet
- Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Strasbourg, France
| | - François Lanza
- Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Strasbourg, France
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23
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Strassel C, Gachet C, Lanza F. On the way to in vitro platelet production. Transfus Clin Biol 2018; 25:220-227. [PMID: 30150135 DOI: 10.1016/j.tracli.2018.07.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2018] [Accepted: 07/16/2018] [Indexed: 02/07/2023]
Abstract
The severely decreased platelet counts (10-30.103 platelets/μL) frequently observed in patients undergoing chemotherapy, radiation treatment or organ transplantation are associated with life-threatening increased bleeding risks. To circumvent these risks, platelet transfusion remains the treatment of choice, despite some limitations which include a limited shelf-life, storage-related deterioration, the development of alloantibodies in recipients and the transmission of infectious diseases. A sustained demand has evolved in recent years for controlled blood products, free of infectious, inflammatory and immune risks. As a consequence, the challenge for blood centers in the near future will be to ensure an adequate supply of blood platelets, which calls for a reassessment of our transfusion models. To meet this challenge, many laboratories are now turning their research efforts towards the in vitro and customized production of blood platelets.
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Affiliation(s)
- Catherine Strassel
- Université de Strasbourg, Inserm, EFS Grand Est, BPPS UMR-S 1255, FMTS, 67000 Strasbourg, France
| | - Christian Gachet
- Université de Strasbourg, Inserm, EFS Grand Est, BPPS UMR-S 1255, FMTS, 67000 Strasbourg, France.
| | - François Lanza
- Université de Strasbourg, Inserm, EFS Grand Est, BPPS UMR-S 1255, FMTS, 67000 Strasbourg, France
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24
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Shepherd JH, Howard D, Waller AK, Foster HR, Mueller A, Moreau T, Evans AL, Arumugam M, Bouët Chalon G, Vriend E, Davidenko N, Ghevaert C, Best SM, Cameron RE. Structurally graduated collagen scaffolds applied to the ex vivo generation of platelets from human pluripotent stem cell-derived megakaryocytes: Enhancing production and purity. Biomaterials 2018; 182:135-144. [PMID: 30118981 DOI: 10.1016/j.biomaterials.2018.08.019] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Revised: 08/03/2018] [Accepted: 08/06/2018] [Indexed: 01/05/2023]
Abstract
Platelet transfusions are a key treatment option for a range of life threatening conditions including cancer, chemotherapy and surgery. Efficient ex vivo systems to generate donor independent platelets in clinically relevant numbers could provide a useful substitute. Large quantities of megakaryocytes (MKs) can be produced from human pluripotent stem cells, but in 2D culture the ratio of platelets harvested from MK cells has been limited and restricts production rate. The development of biomaterial cell supports that replicate vital hematopoietic micro-environment cues are one strategy that may increase in vitro platelet production rates from iPS derived Megakaryocyte cells. In this paper, we present the results obtained generating, simulating and using a novel structurally-graded collagen scaffold within a flow bioreactor system seeded with programmed stem cells. Theoretical analysis of porosity using micro-computed tomography analysis and synthetic micro-particle filtration provided a predictive tool to tailor cell distribution throughout the material. When used with MK programmed stem cells the graded scaffolds influenced cell location while maintaining the ability to continuously release metabolically active CD41 + CD42 + functional platelets. This scaffold design and novel fabrication technique offers a significant advance in understanding the influence of scaffold architectures on cell seeding, retention and platelet production.
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Affiliation(s)
- Jennifer H Shepherd
- Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy, 27 Charles Babbage Road, Cambridge CB3 0FS, UK.
| | - Daniel Howard
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Amie K Waller
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Holly Rebecca Foster
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Annett Mueller
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Thomas Moreau
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Amanda L Evans
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Meera Arumugam
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Guénaëlle Bouët Chalon
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK
| | - Eleonora Vriend
- Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy, 27 Charles Babbage Road, Cambridge CB3 0FS, UK
| | - Natalia Davidenko
- Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy, 27 Charles Babbage Road, Cambridge CB3 0FS, UK
| | - Cedric Ghevaert
- Department of Haematology, University of Cambridge, National Health Blood Service Centre, Long Road, Cambridge CB2 0PT, UK.
| | - Serena M Best
- Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy, 27 Charles Babbage Road, Cambridge CB3 0FS, UK
| | - Ruth E Cameron
- Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy, 27 Charles Babbage Road, Cambridge CB3 0FS, UK
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25
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Vijey P, Posorske B, Machlus KR. In vitro culture of murine megakaryocytes from fetal liver-derived hematopoietic stem cells. Platelets 2018; 29:583-588. [PMID: 30047825 DOI: 10.1080/09537104.2018.1492107] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000-20,000 MKs per liver. Approximately, 20-50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation.
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Affiliation(s)
- Prakrith Vijey
- a Division of Hematology , Brigham and Women's Hospital , Boston , MA , USA
| | - Benjamin Posorske
- a Division of Hematology , Brigham and Women's Hospital , Boston , MA , USA
| | - Kellie R Machlus
- a Division of Hematology , Brigham and Women's Hospital , Boston , MA , USA.,b Department of Medicine , Harvard Medical School , Boston , MA , USA
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26
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Li Y, Jin C, Bai H, Gao Y, Sun S, Chen L, Qin L, Liu PP, Cheng L, Wang QF. Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation. Blood 2018; 131:191-201. [PMID: 29101237 PMCID: PMC5757696 DOI: 10.1182/blood-2017-04-780379] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Accepted: 10/13/2017] [Indexed: 12/19/2022] Open
Abstract
Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.
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Affiliation(s)
- Yueying Li
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
| | - Chen Jin
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Hao Bai
- Division of Hematology, Department of Medicine and
- Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD; and
| | - Yongxing Gao
- Division of Hematology, Department of Medicine and
- Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD; and
| | - Shu Sun
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Lei Chen
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Lei Qin
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Paul P Liu
- Translational and Functional Genomics Branch, National Institutes of Health, National Human Genome Research Institute, Bethesda, MD
| | - Linzhao Cheng
- Division of Hematology, Department of Medicine and
- Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD; and
| | - Qian-Fei Wang
- Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
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27
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Costa MHG, de Soure AM, Cabral JMS, Ferreira FC, da Silva CL. Hematopoietic Niche - Exploring Biomimetic Cues to Improve the Functionality of Hematopoietic Stem/Progenitor Cells. Biotechnol J 2017; 13. [PMID: 29178199 DOI: 10.1002/biot.201700088] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Revised: 10/27/2017] [Indexed: 12/19/2022]
Abstract
The adult bone marrow (BM) niche is a complex entity where a homeostatic hematopoietic system is maintained through a dynamic crosstalk between different cellular and non-cellular players. Signaling mechanisms triggered by cell-cell, cell-extracellular matrix (ECM), cell-cytokine interactions, and local microenvironment parameters are involved in controlling quiescence, self-renewal, differentiation, and migration of hematopoietic stem/progenitor cells (HSPC). A promising strategy to more efficiently expand HSPC numbers and tune their properties ex vivo is to mimic the hematopoietic niche through integration of adjuvant stromal cells, soluble cues, and/or biomaterial-based approaches in HSPC culture systems. Particularly, mesenchymal stem/stromal cells (MSC), through their paracrine activity or direct contact with HSPC, are thought to be a relevant niche player, positioning HSPC-MSC co-culture as a valuable platform to support the ex vivo expansion of hematopoietic progenitors. To improve the clinical outcome of hematopoietic cell transplantation (HCT), namely when the available HSPC are present in a limited number such is the case of HSPC collected from umbilical cord blood (UCB), ex vivo expansion of HSPC is required without eliminating the long-term repopulating capacity of more primitive HSC. Here, we will focus on depicting the characteristics of co-culture systems, as well as other bioengineering approaches to improve the functionality of HSPC ex vivo.
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Affiliation(s)
- Marta H G Costa
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - António M de Soure
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Frederico Castelo Ferreira
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
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28
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Perdomo J, Yan F, Leung HHL, Chong BH. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J Vis Exp 2017. [PMID: 29364213 DOI: 10.3791/56420] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.
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Affiliation(s)
- Jose Perdomo
- Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales;
| | - Feng Yan
- Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales
| | - Halina H L Leung
- Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales
| | - Beng H Chong
- Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales; Haematology Department, St George and Sutherland Hospitals
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29
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Abstract
Ex vivo production of human platelets has been pursued as an alternative measure to resolve limitations in the supply and safety of current platelet transfusion products. To this end, induced pluripotent stem cells (iPSCs) are considered an ideal global source, as they are not only pluripotent and self-renewing, but are also available from basically any person, have relatively few ethical issues, and are easy to manipulate. From human iPSCs, megakaryocyte (MK) lines with robust proliferation capacity have been established by the introduction of specified sets of genes. These expandable MKs are also cryopreservable and thus would be suitable as master cells for good manufacturing practice (GMP)-grade production of platelets, assuring availability on demand and safety against blood-borne infections. Meanwhile, developments in bioreactors that physically mimic the in vivo environment and discovery of substances that promote thrombopoiesis have yielded competent platelets with improved efficiency. The derivation of platelets from iPSCs could further resolve transfusion-related alloimmune complications through the manufacturing of autologous products and human leukocyte antigen (HLA)-compatible platelets from stocked homologous HLA-type iPSC libraries or by manipulation of HLAs and human platelet antigens (HPAs). Considering these key advances in the field, HLA-deleted platelets could become a universal product that is manufactured at industrial level to safely fulfill almost all demands. In this review, we provide an overview of the ex vivo production of iPSC-derived platelets toward clinical applications, a production that would revolutionize the blood transfusion system and lead the field of iPSC-based regenerative medicine.
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Affiliation(s)
- N Sugimoto
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - K Eto
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
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Hodgkinson KM, Kiernan J, Shih AW, Solh Z, Sheffield WP, Pineault N. Intersecting Worlds of Transfusion and Transplantation Medicine: An International Symposium Organized by the Canadian Blood Services Centre for Innovation. Transfus Med Rev 2017; 31:183-192. [DOI: 10.1016/j.tmrv.2017.03.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Revised: 03/17/2017] [Accepted: 03/17/2017] [Indexed: 01/28/2023]
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Zhang C, Neelamegham S. Application of microfluidic devices in studies of thrombosis and hemostasis. Platelets 2017; 28:434-440. [PMID: 28580870 DOI: 10.1080/09537104.2017.1319047] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Due to the importance of fluid flow during thrombotic episodes, it is quite appropriate to study clotting and bleeding processes in devices that have well-defined fluid shear environments. Two common devices for applying these defined shear stresses include the cone-and-plate viscometer and parallel-plate flow chamber. While such tools have many salient features, they require large amounts of blood or other protein components. With growth in the area of microfluidics over the last two decades, it has become feasible to miniaturize such flow devices. Such miniaturization not only enables saving of precious samples but also increases the throughput of fluid shear devices, thus enabling the design of combinatorial experiments and making the technique more accessible to the larger scientific community. In addition to simple flows that are common in traditional flow apparatus, more complex geometries that mimic stenosed arteries and the human microvasculature can also be generated. The composition of the microfluidics cell substrate can also be varied for diverse basic science investigations, and clinical investigations that aim to assay either individual patient coagulopathy or response to anti-coagulation treatment. This review summarizes the current state of the art for such microfluidic devices and their applications in the field of thrombosis and hemostasis.
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Affiliation(s)
- Changjie Zhang
- a Chemical and Biological Engineering, and Clinical & Translational Research Center , University at Buffalo, State University of New York , Buffalo , NY , USA
| | - Sriram Neelamegham
- a Chemical and Biological Engineering, and Clinical & Translational Research Center , University at Buffalo, State University of New York , Buffalo , NY , USA
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Baigger A, Blasczyk R, Figueiredo C. Towards the Manufacture of Megakaryocytes and Platelets for Clinical Application. Transfus Med Hemother 2017. [PMID: 28626367 DOI: 10.1159/000477261] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Platelet transfusions are used in standard clinical practice to prevent hemorrhage in patients suffering from thrombocytopenia or platelet dysfunctions. Recently, a constant rise on the demand of platelets for transfusion has been registered. This may be associated with several factors including demographic changes, population aging as well as incidence and prevalence of hematological diseases. In addition, platelet-regenerative properties have been started to be exploited in different areas such as tissue remodeling and anti-cancer therapies. These new applications are also expected to increase the future demand on platelets. Thus, in vitro generated platelets may constitute a highly desirable alternative to meet the rising demand on platelets. Several factors have been considered in the road trip of producing in vitro megakaryocytes and platelets for clinical application. From selection of the cell source, differentiation protocols and culture conditions to the design of optimal bioreactors, several strategies have been proposed to maximize production yields while preserving functionality. This review summarizes new advances in megakaryocyte and platelet differentiation and their production upscaling.
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Affiliation(s)
- Anja Baigger
- Institute for Transfusion Medicine, Hanover Medical School, Hanover, Germany
| | - Rainer Blasczyk
- Institute for Transfusion Medicine, Hanover Medical School, Hanover, Germany
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Thon JN, Dykstra BJ, Beaulieu LM. Platelet bioreactor: accelerated evolution of design and manufacture. Platelets 2017; 28:472-477. [PMID: 28112988 DOI: 10.1080/09537104.2016.1265922] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Platelets, responsible for clot formation and blood vessel repair, are produced by megakaryocytes in the bone marrow. Platelets are critical for hemostasis and wound healing, and are often provided following surgery, chemotherapy, and major trauma. Despite their importance, platelets today are derived exclusively from human volunteer donors. They have a shelf life of just five days, making platelet shortages common during long weekends, civic holidays, bad weather, and during major emergencies when platelets are needed most. Megakaryocytes in the bone marrow generate platelets by extruding long cytoplasmic extensions called proplatelets through gaps/fenestrations in blood vessels. Proplatelets serve as assembly lines for platelet production by sequentially releasing platelets and large discoid-shaped platelet intermediates called preplatelets into the circulation. Recent advances in platelet bioreactor development have aimed to mimic the key physiological characteristics of bone marrow, including extracellular matrix composition/stiffness, blood vessel architecture comprising tissue-specific microvascular endothelium, and shear stress. Nevertheless, how complex interactions within three-dimensional (3D) microenvironments regulate thrombopoiesis remains poorly understood, and the technical challenges associated with designing and manufacturing biomimetic microfluidic devices are often under-appreciated and under-reported. We have previously reviewed the major cell culture, platelet quality assessment, and regulatory roadblocks that must be overcome to make human platelet production possible for clinical use [1]. This review builds on our previous manuscript by: (1) detailing the historical evolution of platelet bioreactor design to recapitulate native platelet production ex vivo, and (2) identifying the associated challenges that still need to be addressed to further scale and validate these devices for commercial application. While platelets are among the first cells whose ex vivo production is spearheading major engineering advancements in microfluidic design, the resulting discoveries will undoubtedly extend to the production of other human tissues. This work is critical to identify the physiological characteristics of relevant 3D tissue-specific microenvironments that drive cell differentiation and elaborate upon how these are disrupted in disease. This is a burgeoning field whose future will define not only the ex vivo production of platelets and development of targeted therapies for thrombocytopenia, but the promise of regenerative medicine for the next century.
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Affiliation(s)
- Jonathan N Thon
- a Hematology Division, Department of Medicine , Brigham and Women's Hospital , MA , USA.,b Harvard Medical School , Boston , MA , USA.,c Platelet BioGenesis , Boston , MA , USA
| | - Brad J Dykstra
- a Hematology Division, Department of Medicine , Brigham and Women's Hospital , MA , USA.,b Harvard Medical School , Boston , MA , USA.,c Platelet BioGenesis , Boston , MA , USA
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Heazlewood SY, Nilsson SK, Cartledge K, Be CL, Vinson A, Gel M, Haylock DN. Progress in bio-manufacture of platelets for transfusion. Platelets 2017; 28:649-656. [DOI: 10.1080/09537104.2016.1257783] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Affiliation(s)
- Shen Y. Heazlewood
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
- The Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
| | - Susan K. Nilsson
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
- The Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
| | - Kellie Cartledge
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
| | - Cheang Ly Be
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
| | - Andrew Vinson
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
- The Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
| | - Murat Gel
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
| | - David N. Haylock
- Manufacturing, Commonwealth Scientific Industrial Research Organisation, Clayton, Australia
- The Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
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Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets. Blood 2016; 127:2231-40. [PMID: 26966088 DOI: 10.1182/blood-2015-09-670208] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 03/04/2016] [Indexed: 12/29/2022] Open
Abstract
The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production.
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Nelson MR, Roy K. Bone-marrow mimicking biomaterial niches for studying hematopoietic stem and progenitor cells. J Mater Chem B 2016; 4:3490-3503. [DOI: 10.1039/c5tb02644j] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
This review discusses the considerations and approaches that have been employed for designing biomaterial based cultures for replicating the hematopoietic stem and progenitor cell niche.
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Affiliation(s)
- Michael R. Nelson
- Wallace H. Coulter Department of Biomedical Engineering at the Georgia Tech and Emory University
- The Parker H. Petit Institute for Bioengineering and Biosciences
- Georgia Institute of Technology
- Atlanta
- USA
| | - Krishnendu Roy
- Wallace H. Coulter Department of Biomedical Engineering at the Georgia Tech and Emory University
- The Parker H. Petit Institute for Bioengineering and Biosciences
- Georgia Institute of Technology
- Atlanta
- USA
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Yang Y, Liu C, Lei X, Wang H, Su P, Ru Y, Ruan X, Duan E, Feng S, Han M, Xu Y, Shi L, Jiang E, Zhou J. Integrated Biophysical and Biochemical Signals Augment Megakaryopoiesis and Thrombopoiesis in a Three-Dimensional Rotary Culture System. Stem Cells Transl Med 2015; 5:175-85. [PMID: 26702125 DOI: 10.5966/sctm.2015-0080] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Accepted: 10/12/2015] [Indexed: 12/22/2022] Open
Abstract
Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of the platelet supply limits the care of patients. Although derivation of clinical-scale platelets in vitro could provide a new source for transfusion, the devices and procedures for deriving scalable platelets for clinical applications have not been established. In the present study, we found that a rotary cell culture system (RCCS) can potentiate megakaryopoiesis and significantly improve the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the RCCS improved platelet generation efficiency by as much as ∼3.7-fold compared with static conditions. Shear force, simulated microgravity, and better diffusion of nutrients and oxygen from the RCCS, altogether, might account for the improved efficient platelet generation. The cost-effective and highly controllable strategy and methodology represent an important step toward large-scale platelet production for future biomedical and clinical applications. Significance: Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of platelet supply limits the care of patients. Thus, derivation of clinical-scale platelets in vitro would provide a new source for transfusion. The present study evaluated a rotary suspension cell culture system that was able to potentiate megakaryopoiesis and significantly improved the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the three-dimensional system improved platelet generation efficiency compared with the static condition. The three-dimensional device and the strategy developed in the present study should markedly improve the generation of large-scale platelets for use in future biomedical and clinical settings.
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Affiliation(s)
- Yiqing Yang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China Faculty of Laboratory Medical Science, Hebei North University, Zhangjiakou, People's Republic of China
| | - CuiCui Liu
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Xiaohua Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, Beijing, People's Republic of China
| | - Hongtao Wang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Pei Su
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Yongxin Ru
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Xinhua Ruan
- Department of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin, People's Republic of China
| | - Enkui Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, Beijing, People's Republic of China
| | - Sizhou Feng
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Mingzhe Han
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Yuanfu Xu
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Lihong Shi
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Erlie Jiang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Jiaxi Zhou
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
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Yang Y, Liu C, Lei X, Wang H, Su P, Ru Y, Ruan X, Duan E, Feng S, Han M, Xu Y, Shi L, Jiang E, Zhou J. Integrated Biophysical and Biochemical Signals Augment Megakaryopoiesis and Thrombopoiesis in a Three-Dimensional Rotary Culture System. Stem Cells Transl Med 2015. [DOI: dx.doi.org/10.5966/sctm.2015-0080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Abstract
Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of the platelet supply limits the care of patients. Although derivation of clinical-scale platelets in vitro could provide a new source for transfusion, the devices and procedures for deriving scalable platelets for clinical applications have not been established. In the present study, we found that a rotary cell culture system (RCCS) can potentiate megakaryopoiesis and significantly improve the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the RCCS improved platelet generation efficiency by as much as ∼3.7-fold compared with static conditions. Shear force, simulated microgravity, and better diffusion of nutrients and oxygen from the RCCS, altogether, might account for the improved efficient platelet generation. The cost-effective and highly controllable strategy and methodology represent an important step toward large-scale platelet production for future biomedical and clinical applications.
Significance
Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of platelet supply limits the care of patients. Thus, derivation of clinical-scale platelets in vitro would provide a new source for transfusion. The present study evaluated a rotary suspension cell culture system that was able to potentiate megakaryopoiesis and significantly improved the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the three-dimensional system improved platelet generation efficiency compared with the static condition. The three-dimensional device and the strategy developed in the present study should markedly improve the generation of large-scale platelets for use in future biomedical and clinical settings.
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Affiliation(s)
- Yiqing Yang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
- Faculty of Laboratory Medical Science, Hebei North University, Zhangjiakou, People's Republic of China
| | - CuiCui Liu
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Xiaohua Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, Beijing, People's Republic of China
| | - Hongtao Wang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Pei Su
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Yongxin Ru
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Xinhua Ruan
- Department of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin, People's Republic of China
| | - Enkui Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, Beijing, People's Republic of China
| | - Sizhou Feng
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Mingzhe Han
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Yuanfu Xu
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Lihong Shi
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Erlie Jiang
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
| | - Jiaxi Zhou
- State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China
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Avanzi MP, Oluwadara OE, Cushing MM, Mitchell ML, Fischer S, Mitchell WB. A novel bioreactor and culture method drives high yields of platelets from stem cells. Transfusion 2015; 56:170-8. [DOI: 10.1111/trf.13375] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Revised: 07/10/2015] [Accepted: 07/16/2015] [Indexed: 12/26/2022]
Affiliation(s)
| | | | | | | | | | - W. Beau Mitchell
- New York Blood Center; New York New York
- Weill Cornell Medical College; New York New York
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40
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Balduini A, Di Buduo CA, Kaplan DL. Translational approaches to functional platelet production ex vivo. Thromb Haemost 2015; 115:250-6. [PMID: 26353819 DOI: 10.1160/th15-07-0570] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Accepted: 08/11/2015] [Indexed: 12/13/2022]
Abstract
Platelets, which are released by megakaryocytes, play key roles in haemostasis, angiogenesis, immunity, tissue regeneration and wound healing. The scarcity of clinical cures for life threatening platelet diseases is in a large part due to limited insight into the mechanisms that control the developmental process of megakaryocytes and the mechanisms that govern the production of platelets within the bone marrow. To overcome these limitations, functional human tissue models have been developed and studied to extrapolate ex vivo outcomes for new insight on bone marrow functions in vivo. There are many challenges that these models must overcome, from faithfully mimicking the physiological composition and functions of bone marrow, to the collection of the platelets generated and validation of their viability and function for human use. The overall goal is to identify innovative instruments to study mechanisms of platelet release, diseases related to platelet production and new therapeutic targets starting from human progenitor cells.
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Affiliation(s)
- Alessandra Balduini
- Alessandra Balduini, Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA, Tel.: +1 617 627 2580, Fax: +1 617 627 3231, E-mail:
| | | | - David L Kaplan
- David L. Kaplan, Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA, Tel.: +1 617 627 2580, Fax: +1 617 627 3231, E-mail:
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Pietrzyk-Nivau A, Poirault-Chassac S, Gandrille S, Derkaoui SM, Kauskot A, Letourneur D, Le Visage C, Baruch D. Three-Dimensional Environment Sustains Hematopoietic Stem Cell Differentiation into Platelet-Producing Megakaryocytes. PLoS One 2015; 10:e0136652. [PMID: 26313154 PMCID: PMC4552162 DOI: 10.1371/journal.pone.0136652] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2014] [Accepted: 08/05/2015] [Indexed: 11/18/2022] Open
Abstract
Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.
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Affiliation(s)
| | | | - Sophie Gandrille
- INSERM, UMR-S 1140, University Paris Descartes, Sorbonne Paris Cité, Paris, France
- AP-HP, Georges Pompidou European Hospital, Department of Hematology, Paris, France
| | - Sidi-Mohammed Derkaoui
- INSERM, UMR-S 1148, University Paris Diderot, Paris; University Paris Nord, Villetaneuse, Sorbonne Paris Cité, France
| | - Alexandre Kauskot
- INSERM, UMR-S 1140, University Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Didier Letourneur
- INSERM, UMR-S 1148, University Paris Diderot, Paris; University Paris Nord, Villetaneuse, Sorbonne Paris Cité, France
| | - Catherine Le Visage
- INSERM, UMR-S 1148, University Paris Diderot, Paris; University Paris Nord, Villetaneuse, Sorbonne Paris Cité, France
| | - Dominique Baruch
- INSERM, UMR-S 1140, University Paris Descartes, Sorbonne Paris Cité, Paris, France
- * E-mail:
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Kamat V, Muthard RW, Li R, Diamond SL. Microfluidic assessment of functional culture-derived platelets in human thrombi under flow. Exp Hematol 2015; 43:891-900.e4. [PMID: 26145051 DOI: 10.1016/j.exphem.2015.06.302] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2015] [Revised: 06/12/2015] [Accepted: 06/25/2015] [Indexed: 11/30/2022]
Abstract
Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 μL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow.
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Affiliation(s)
- Viraj Kamat
- Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Ryan W Muthard
- Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Ruizhi Li
- Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Scott L Diamond
- Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania.
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44
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Abstract
Historically, platelet transfusion has proven a reliable way to treat patients suffering from thrombocytopenia or similar ailments. An undersupply of donors, however, has demanded alternative platelet sources. Scientists have therefore sought to recapitulate the biological events that convert hematopoietic stem cells into platelets in the laboratory. Such platelets have shown good function and potential for treatment. Yet the number manufactured ex vivo falls well short of clinical application. Part of the reason is the remarkable gaps in our understanding of the molecular mechanisms driving platelet formation. Using several stem cell sources, scientists have progressively clarified the chemical signaling and physical microenvironment that optimize ex vivo platelets and reconstituted them in synthetic environments. Key advances in cell reprogramming and the ability to propagate self-renewal have extended the lifetime of megakaryocytes to increase the pool of platelet progenitors.
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Affiliation(s)
- P Karagiannis
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto, Japan
| | - K Eto
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto, Japan
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45
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Thon JN, Medvetz DA, Karlsson SM, Italiano JE. Road blocks in making platelets for transfusion. J Thromb Haemost 2015; 13 Suppl 1:S55-62. [PMID: 26149051 PMCID: PMC5565795 DOI: 10.1111/jth.12942] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The production of laboratory-generated human platelets is necessary to meet present and future transfusion needs. This manuscript will identify and define the major roadblocks that must be overcome to make human platelet production possible for clinical use, and propose solutions necessary to accelerate development of laboratory-generated human platelets to market.
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Affiliation(s)
- J N Thon
- Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
- Platelet BioGenesis, Chestnut Hill, MA, USA
| | - D A Medvetz
- Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | | | - J E Italiano
- Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
- Platelet BioGenesis, Chestnut Hill, MA, USA
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46
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Pineault N, Boisjoli GJ. Megakaryopoiesis andex vivodifferentiation of stem cells into megakaryocytes and platelets. ACTA ACUST UNITED AC 2015. [DOI: 10.1111/voxs.12155] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- N. Pineault
- Center for Innovation; Canadian Blood Services; Ottawa ON Canada
- Department of Biochemistry, Microbiology and Immunology; University of Ottawa; Ottawa ON Canada
| | - G. J. Boisjoli
- Center for Innovation; Canadian Blood Services; Ottawa ON Canada
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47
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Di Buduo CA, Wray LS, Tozzi L, Malara A, Chen Y, Ghezzi CE, Smoot D, Sfara C, Antonelli A, Spedden E, Bruni G, Staii C, De Marco L, Magnani M, Kaplan DL, Balduini A. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood 2015; 125:2254-64. [PMID: 25575540 PMCID: PMC4383799 DOI: 10.1182/blood-2014-08-595561] [Citation(s) in RCA: 122] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Accepted: 01/03/2015] [Indexed: 01/16/2023] Open
Abstract
We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.
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Affiliation(s)
- Christian A Di Buduo
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Biotechnology Research Laboratories, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy
| | - Lindsay S Wray
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Biotechnology Research Laboratories, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy; Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Lorenzo Tozzi
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Biotechnology Research Laboratories, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy; Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Alessandro Malara
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Biotechnology Research Laboratories, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy
| | - Ying Chen
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Chiara E Ghezzi
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Daniel Smoot
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Carla Sfara
- Department of Biomolecular Sciences, Biochemistry and Molecular Biology Section, University of Urbino "Carlo Bo," Urbino, Italy
| | - Antonella Antonelli
- Department of Biomolecular Sciences, Biochemistry and Molecular Biology Section, University of Urbino "Carlo Bo," Urbino, Italy
| | - Elise Spedden
- Department of Physics, Tufts University, Medford, MA
| | - Giovanna Bruni
- Department of Chemistry, Physical Chemistry Section, University of Pavia, Pavia, Italy
| | | | - Luigi De Marco
- Department of Translational Research, Stem Cells Unit, Istituto di Ricovero e Cura a Carattere Scientifico Centro di Riferimento Oncologico, Aviano, Italy; and Department of Molecular and Experimental Research, The Scripps Research Institute, La Jolla, CA
| | - Mauro Magnani
- Department of Biomolecular Sciences, Biochemistry and Molecular Biology Section, University of Urbino "Carlo Bo," Urbino, Italy
| | - David L Kaplan
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Alessandra Balduini
- Department of Molecular Medicine, University of Pavia, Pavia, Italy; Biotechnology Research Laboratories, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy; Department of Biomedical Engineering, Tufts University, Medford, MA
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Malara A, Abbonante V, Di Buduo CA, Tozzi L, Currao M, Balduini A. The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control. Cell Mol Life Sci 2015; 72:1517-36. [PMID: 25572292 PMCID: PMC4369169 DOI: 10.1007/s00018-014-1813-y] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2014] [Revised: 12/15/2014] [Accepted: 12/19/2014] [Indexed: 12/19/2022]
Abstract
Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.
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Affiliation(s)
- Alessandro Malara
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia, Italy
| | - Vittorio Abbonante
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia, Italy
| | - Christian A. Di Buduo
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia, Italy
| | - Lorenzo Tozzi
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Department of Biomedical Engineering, Tufts University, Medford, MA USA
| | - Manuela Currao
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia, Italy
| | - Alessandra Balduini
- Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
- Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia, Italy
- Department of Biomedical Engineering, Tufts University, Medford, MA USA
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49
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Hatami J, Andrade PZ, Alves de Matos AP, Djokovic D, Lilaia C, Ferreira FC, Cabral JMS, da Silva CL. Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells. Cytotherapy 2015; 17:428-42. [PMID: 25680300 DOI: 10.1016/j.jcyt.2014.12.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 12/18/2014] [Accepted: 12/23/2014] [Indexed: 12/12/2022]
Abstract
BACKGROUND AIMS Platelet transfusion can be a life-saving procedure in different medical settings. Thus, there is an increasing demand for platelets, of which shelf-life is only 5 days. The efficient ex vivo biomanufacturing of platelets would allow overcoming the shortages of donated platelets. METHODS We exploited a two-stage culture protocol aiming to study the effect of different parameters on the megakaryo/thrombopoiesis ex vivo. In the expansion stage, human umbilical cord blood (UCB)-derived CD34(+)-enriched cells were expanded in co-culture with human bone marrow mesenchymal stromal cells (BM-MSCs). The megakaryocytic commitment and platelet generation were studied, considering the impact of exogenous addition of thrombopoietin (TPO) in the expansion stage and a cytokine cocktail (Cyt) including TPO and interleukin-3 in the differentiation stage, with the use of different culture medium formulations, and in the presence/absence of BM-MSCs (direct versus non-direct cell-cell contact). RESULTS Our results suggest that an early megakaryocytic commitment, driven by TPO addition during the expansion stage, further enhanced megakaryopoiesis. Importantly, the results suggest that co-culture with BM-MSCs under serum-free conditions combined with Cyt addition, in the differentiation stage, significantly improved the efficiency yield of megakaryo/thrombopoiesis as well as increasing %CD41, %CD42b and polyploid content; in particular, direct contact of expanded cells with BM-MSCs, in the differentiation stage, enhanced the efficiency yield of megakaryo/thrombopoiesis, despite inhibiting their maturation. CONCLUSIONS The present study established an in vitro model for the hematopoietic niche that combines different biological factors, namely, the presence of stromal/accessory cells and biochemical cues, which mimics the BM niche and enhances an efficient megakaryo/thrombopoiesis process ex vivo.
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Affiliation(s)
- Javad Hatami
- Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Pedro Z Andrade
- Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - António Pedro Alves de Matos
- Centro de Estudos do Ambiente e do Mar (CESAM/FCUL)-Faculdade de Ciências da Universidade de Lisboa and Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Campus Universitário, Quinta da Granja, Monte de Caparica, Caparica, Portugal
| | - Dusan Djokovic
- Department of Obstetrics, Centro Hospitalar Lisboa Ocidental E.P.E., Hospital São Francisco Xavier, Lisboa, Portugal
| | - Carla Lilaia
- Department of Obstetrics, Centro Hospitalar Lisboa Ocidental E.P.E., Hospital São Francisco Xavier, Lisboa, Portugal
| | - Frederico Castelo Ferreira
- Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
| | - Joaquim M S Cabral
- Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
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50
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Harada T, Hirabayashi Y, Hatta Y, Tsuboi I, Glomm WR, Yasuda M, Aizawa S. Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system. Growth Factors 2015; 33:347-55. [PMID: 26431462 DOI: 10.3109/08977194.2015.1088534] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and "quietly" regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell-stromal cell interactions in vitro.
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Affiliation(s)
| | - Yukio Hirabayashi
- a Department of Functional Morphology and
- b Department of Medicine , Nihon University School of Medicine , Tokyo , Japan
| | - Yoshihiro Hatta
- b Department of Medicine , Nihon University School of Medicine , Tokyo , Japan
| | | | - Wilhelm Robert Glomm
- c Department of Chemical Engineering , Norwegian University of Science and Technology , Trondheim , Norway , and
| | - Masahiro Yasuda
- d Department of Chemical Engineering , Osaka Prefecture University , Osaka , Japan
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