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1
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Palis J. Erythropoiesis in the mammalian embryo. Exp Hematol 2024; 136:104283. [PMID: 39048071 DOI: 10.1016/j.exphem.2024.104283] [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: 07/03/2024] [Revised: 07/11/2024] [Accepted: 07/15/2024] [Indexed: 07/27/2024]
Abstract
Red blood cells (RBCs) comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the developing mammalian embryo. Examination of circulating blood cells in mammalian embryos revealed two distinct types of erythroid cells: large, nucleated "primitive" erythroblasts followed by smaller, enucleated "definitive" erythrocytes. This review describes the current understanding of primitive and definitive erythropoiesis gleaned from studies of mouse and human embryos and induced pluripotent stem cells (iPSCs). Primitive erythropoiesis in the mouse embryo comprises a transient wave of committed primitive erythroid progenitors (primitive erythroid colony-forming cells, EryP-CFC) in the early yolk sac that generates a robust cohort of precursors that mature in the bloodstream and enucleate. In contrast, definitive erythropoiesis has two distinct developmental origins. The first comprises a transient wave of definitive erythroid progenitors (burst-forming units erythroid, BFU-E) that emerge in the yolk sac and seed the fetal liver where they terminally mature to provide the first definitive RBCs. The second comprises hematopoietic stem cell (HSC)-derived BFU-E that terminally mature at sites colonized by HSCs particularly the fetal liver and subsequently the bone marrow. Primitive and definitive erythropoiesis are derived from endothelial identity precursors with distinct developmental origins. Although they share prototypical transcriptional regulation, primitive and definitive erythropoiesis are also characterized by distinct lineage-specific factors. The exquisitely timed, sequential production of primitive and definitive erythroid cells is necessary for the survival and growth of the mammalian embryo.
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Affiliation(s)
- James Palis
- Department of Pediatrics, University of Rochester Medical Center, Rochester, NY.
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2
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Ju H, Sohn Y, Nam Y, Rim YA. Progresses in overcoming the limitations of in vitro erythropoiesis using human induced pluripotent stem cells. Stem Cell Res Ther 2024; 15:142. [PMID: 38750578 PMCID: PMC11094930 DOI: 10.1186/s13287-024-03754-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2024] [Accepted: 05/04/2024] [Indexed: 05/19/2024] Open
Abstract
Researchers have attempted to generate transfusable oxygen carriers to mitigate RBC supply shortages. In vitro generation of RBCs using stem cells such as hematopoietic stem and progenitor cells (HSPCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) has shown promise. Specifically, the limited supplies of HSPCs and ethical issues with ESCs make iPSCs the most promising candidate for in vitro RBC generation. However, researchers have encountered some major challenges when using iPSCs to produce transfusable RBC products, such as enucleation and RBC maturation. In addition, it has proven difficult to manufacture these products on a large scale. In this review, we provide a brief overview of erythropoiesis and examine endeavors to recapitulate erythropoiesis in vitro using various cell sources. Furthermore, we explore the current obstacles and potential solutions aimed at enabling the large-scale production of transfusable RBCs in vitro.
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Affiliation(s)
- Hyeonwoo Ju
- Department of Biotechnology, Yonsei University, Seoul, 03722, Korea
| | - Yeowon Sohn
- Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, South Korea
| | - Yoojun Nam
- Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, South Korea.
- YiPSCELL Inc., L2 Omnibus Park, Banpo-dearo 222, Seocho-gu, Seoul, 06591, Republic of Korea.
| | - Yeri Alice Rim
- YiPSCELL Inc., L2 Omnibus Park, Banpo-dearo 222, Seocho-gu, Seoul, 06591, Republic of Korea.
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 06591, Republic of Korea.
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3
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Ma L, Wu Q, Tam PKH. The Current Proceedings of PSC-Based Liver Fibrosis Therapy. Stem Cell Rev Rep 2023; 19:2155-2165. [PMID: 37490204 DOI: 10.1007/s12015-023-10592-4] [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] [Accepted: 07/12/2023] [Indexed: 07/26/2023]
Abstract
Liver fibrosis was initially considered to be an irreversible process which will eventually lead to the occurrence of liver cancer. So far there has been no effective therapeutic approach to treat liver fibrosis although scientists have put tremendous efforts into the underlying mechanisms of this disease. Therefore, in-depth research on novel and safe treatments of liver fibrosis is of great significance to human health. Pluripotent stem cells (PSCs) play important roles in the study of liver fibrosis due to their unique features in self-renewal ability, pluripotency, and paracrine function. This article mainly reviews the applications of PSCs in the study of liver fibrosis in recent years. We discuss the role of PSC-derived liver organoids in the study of liver fibrosis, and the latest research advances on the differentiation of PSCs into hepatocytes or macrophages. We also highlight the importance of exosomes of PSCs for the treatment of liver fibrosis.
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Affiliation(s)
- Li Ma
- The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, China
| | - Qiang Wu
- The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, China.
| | - Paul Kwong-Hang Tam
- Faculty of Medicine, Macau University of Science and Technology, Taipa, China.
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4
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Lee SJ, Jung C, Oh JE, Kim S, Lee S, Lee JY, Yoon YS. Generation of Red Blood Cells from Human Pluripotent Stem Cells-An Update. Cells 2023; 12:1554. [PMID: 37296674 PMCID: PMC10253210 DOI: 10.3390/cells12111554] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2023] [Revised: 05/31/2023] [Accepted: 06/02/2023] [Indexed: 06/12/2023] Open
Abstract
Red blood cell (RBC) transfusion is a lifesaving medical procedure that can treat patients with anemia and hemoglobin disorders. However, the shortage of blood supply and risks of transfusion-transmitted infection and immune incompatibility present a challenge for transfusion. The in vitro generation of RBCs or erythrocytes holds great promise for transfusion medicine and novel cell-based therapies. While hematopoietic stem cells and progenitors derived from peripheral blood, cord blood, and bone marrow can give rise to erythrocytes, the use of human pluripotent stem cells (hPSCs) has also provided an important opportunity to obtain erythrocytes. These hPSCs include both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). As hESCs carry ethical and political controversies, hiPSCs can be a more universal source for RBC generation. In this review, we first discuss the key concepts and mechanisms of erythropoiesis. Thereafter, we summarize different methodologies to differentiate hPSCs into erythrocytes with an emphasis on the key features of human definitive erythroid lineage cells. Finally, we address the current limitations and future directions of clinical applications using hiPSC-derived erythrocytes.
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Affiliation(s)
- Shin-Jeong Lee
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
- Research and Development Center, KarisBio Inc., 50-1 Yonsei-Ro, Avison Biomedical Research Center Room 525, Seodaemun-gu, Seoul 03722, Republic of Korea
| | - Cholomi Jung
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
- Department of Internal Medicine, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Jee Eun Oh
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
- Research and Development Center, KarisBio Inc., 50-1 Yonsei-Ro, Avison Biomedical Research Center Room 525, Seodaemun-gu, Seoul 03722, Republic of Korea
| | - Sangsung Kim
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
- Research and Development Center, KarisBio Inc., 50-1 Yonsei-Ro, Avison Biomedical Research Center Room 525, Seodaemun-gu, Seoul 03722, Republic of Korea
| | - Sangho Lee
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA;
| | - Ji Yoon Lee
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
| | - Young-sup Yoon
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; (S.-J.L.); (C.J.); (J.E.O.); (S.K.)
- Research and Development Center, KarisBio Inc., 50-1 Yonsei-Ro, Avison Biomedical Research Center Room 525, Seodaemun-gu, Seoul 03722, Republic of Korea
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA;
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5
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Ding J, Li Y, Larochelle A. De Novo Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells for Cellular Therapy. Cells 2023; 12:321. [PMID: 36672255 PMCID: PMC9857267 DOI: 10.3390/cells12020321] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 01/02/2023] [Accepted: 01/11/2023] [Indexed: 01/19/2023] Open
Abstract
The ability to manufacture human hematopoietic stem cells (HSCs) in the laboratory holds enormous promise for cellular therapy of human blood diseases. Several differentiation protocols have been developed to facilitate the emergence of HSCs from human pluripotent stem cells (PSCs). Most approaches employ a stepwise addition of cytokines and morphogens to recapitulate the natural developmental process. However, these protocols globally lack clinical relevance and uniformly induce PSCs to produce hematopoietic progenitors with embryonic features and limited engraftment and differentiation capabilities. This review examines how key intrinsic cues and extrinsic environmental inputs have been integrated within human PSC differentiation protocols to enhance the emergence of definitive hematopoiesis and how advances in genomics set the stage for imminent breakthroughs in this field.
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Affiliation(s)
| | | | - Andre Larochelle
- Cellular and Molecular Therapeutics Branch, National Heart Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
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6
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Fidanza A, Forrester LM. Progress in the production of haematopoietic stem and progenitor cells from human pluripotent stem cells. ACTA ACUST UNITED AC 2021; 13:100050. [PMID: 34405125 PMCID: PMC8350141 DOI: 10.1016/j.regen.2021.100050] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Revised: 06/14/2021] [Accepted: 06/21/2021] [Indexed: 12/16/2022]
Abstract
Cell therapies are currently used to treat many haematological diseases. These treatments range from the long-term reconstitution of the entire haematopoietic system using the most potent haematopoietic stem cells (HSCs) to the short-term rescue with mature functional end cells such as oxygen-carrying red blood cells and cells of the immune system that can fight infection and repair tissue. Limitations in supply and the risk of transmitting infection has prompted the design of protocols to produce some of these cell types from human pluripotent stem cells (hPSCs). Although it has proven challenging to generate the most potent HSCs directly from hPSCs, significant progress has been made in the development of differentiation protocols that can successfully produce haematopoietic progenitor cells and most of the mature cell lineages. We review the key steps used in the production of haematopoietic stem and progenitor cells (HSPCs) from hPSCs and the cell surface markers and reporter strategies that have been used to define specific transitions. Most studies have relied on the use of known markers that define HSPC production in vivo but more recently single cell RNA sequencing has allowed a less biased approach to their characterisation. Transcriptional profiling has identified new markers for naïve and committed hPSC-derived HSPC populations and trajectory analyses has provided novel insights into their lineage potential. Direct comparison of in vitro- and in vivo-derived RNA single cell sequencing datasets has highlights similarities and differences between the two systems and this deeper understanding will be key to the design and the tracking of improved and more efficient differentiation protocols.
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Affiliation(s)
- Antonella Fidanza
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, UK
| | - Lesley M Forrester
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, UK
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7
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Viswanathan P, Gupta P, Sharma Y, Maisuradze L, Bandi S, Gupta S. Caffeine disrupts ataxia telangiectasia mutated gene-related pathways and exacerbates acetaminophen toxicity in human fetal immortalized hepatocytes. Toxicology 2021; 457:152811. [PMID: 33971260 DOI: 10.1016/j.tox.2021.152811] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 04/24/2021] [Accepted: 05/04/2021] [Indexed: 11/27/2022]
Abstract
Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.
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Affiliation(s)
- Preeti Viswanathan
- Division of Pediatric Gastroenterology and Department of Pediatrics, Children's Hospital at Montefiore, USA
| | | | | | | | - Sriram Bandi
- Department of Medicine, USA; Marion Bessin Liver Research Center, USA
| | - Sanjeev Gupta
- Department of Medicine, USA; Marion Bessin Liver Research Center, USA; Department of Pathology, USA; Diabetes Center, USA; Fleischer Institute for Diabetes and Metabolism, USA; Irwin S. and Sylvia Chanin Institute for Cancer Research, USA; Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York, USA.
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8
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Ebrahimi M, Forouzesh M, Raoufi S, Ramazii M, Ghaedrahmati F, Farzaneh M. Differentiation of human induced pluripotent stem cells into erythroid cells. Stem Cell Res Ther 2020; 11:483. [PMID: 33198819 PMCID: PMC7667818 DOI: 10.1186/s13287-020-01998-9] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 10/25/2020] [Indexed: 02/07/2023] Open
Abstract
During the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.
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Affiliation(s)
- Mohsen Ebrahimi
- Neonatal and Children's Health Research Center, Golestan University of Medical Sciences, Gorgan, Iran
| | - Mehdi Forouzesh
- Legal Medicine Organization of Iran, Legal Medicine Research Center, Legal Medicine organization, Tehran, Iran
| | - Setareh Raoufi
- Faculty of Medical Sciences and Technologies, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Mohammad Ramazii
- Kerman University of Medical Sciences, University of Kerman, Kerman, Iran
| | - Farhoodeh Ghaedrahmati
- Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Maryam Farzaneh
- Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
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9
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The erythroblastic island niche: modeling in health, stress, and disease. Exp Hematol 2020; 91:10-21. [DOI: 10.1016/j.exphem.2020.09.185] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 09/02/2020] [Accepted: 09/03/2020] [Indexed: 12/19/2022]
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10
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Fibach E. Erythropoiesis In Vitro-A Research and Therapeutic Tool in Thalassemia. J Clin Med 2019; 8:jcm8122124. [PMID: 31810354 PMCID: PMC6947291 DOI: 10.3390/jcm8122124] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2019] [Revised: 11/26/2019] [Accepted: 11/29/2019] [Indexed: 12/13/2022] Open
Abstract
Thalassemia (thal) is a hereditary chronic hemolytic anemia due to a partial or complete deficiency in the production of globin chains, in most cases, α or β, which compose, together with the iron-containing porphyrins (hemes), the hemoglobin molecules in red blood cells (RBC). The major clinical symptom of β-thal is severe chronic anemia—a decrease in RBC number and their hemoglobin content. In spite of the improvement in therapy, thal still severely affects the quality of life of the patients and their families and imposes a substantial financial burden on the community. These considerations position β-thal, among other hemoglobinopathies, as a major health and social problem that deserves increased efforts in research and its clinical application. These efforts are based on clinical studies, experiments in animal models and the use of erythroid cells grown in culture. The latter include immortal cell lines and cultures initiated by erythroid progenitor and stem cells derived from the blood and RBC producing (erythropoietic) sites of normal and thal donors, embryonic stem cells, and recently, "induced pluripotent stem cells" generated by manipulation of differentiated somatic cells. The present review summarizes the use of erythroid cultures, their technological aspects and their contribution to the research and its clinical application in thal. The former includes deciphering of the normal and pathological biology of the erythroid cell development, and the latter—their role in developing innovative therapeutics—drugs and methods of gene therapy, as well as providing an alternative source of RBC that may complement or substitute blood transfusions.
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Affiliation(s)
- Eitan Fibach
- The Hematology Department, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel
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11
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Differentiation of Baboon ( Papio anubis) Induced-Pluripotent Stem Cells into Enucleated Red Blood Cells. Cells 2019; 8:cells8101282. [PMID: 31635069 PMCID: PMC6829891 DOI: 10.3390/cells8101282] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Revised: 10/04/2019] [Accepted: 10/16/2019] [Indexed: 01/14/2023] Open
Abstract
As cell culture methods and stem cell biology have progressed, the in vitro production of cultured RBCs (cRBCs) has emerged as a viable option to produce cells for transfusion or to carry therapeutic cargoes. RBCs produced in culture can be quality-tested either by xeno-transfusion of human cells into immuno-deficient animals, or by transfusion of autologous cells in immuno-competent models. Although murine xeno-transfusion methods have improved, they must be complemented by studies in immuno-competent models. Non-human primates (NHPs) are important pre-clinical, large animal models due to their high biological and developmental similarities with humans, including their comparable hematopoietic and immune systems. Among NHPs, baboons are particularly attractive to validate cRBCs because of the wealth of data available on the characteristics of RBCs in this species that have been generated by past blood transfusion studies. We report here that we have developed a method to produce enucleated cRBCs by differentiation of baboon induced pluripotent stem cells (iPSCs). This method will enable the use of baboons to evaluate therapeutic cRBCs and generate essential pre-clinical data in an immuno-competent, large animal model. Production of the enucleated baboon cRBCs was achieved by adapting the PSC-RED protocol that we previously developed for human cells. Baboon-PSC-RED is an efficient chemically-defined method to differentiate iPSCs into cRBCs that are about 40% to 50% enucleated. PSC-RED is relatively low cost because it requires no albumin and only small amounts of recombinant transferrin.
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12
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Olivier EN, Zhang S, Yan Z, Suzuka S, Roberts K, Wang K, Bouhassira EE. PSC-RED and MNC-RED: Albumin-free and low-transferrin robust erythroid differentiation protocols to produce human enucleated red blood cells. Exp Hematol 2019; 75:31-52.e15. [PMID: 31176681 DOI: 10.1016/j.exphem.2019.05.006] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2019] [Revised: 05/23/2019] [Accepted: 05/24/2019] [Indexed: 01/19/2023]
Abstract
Many methods have been developed to produce cultured red blood cells (cRBCs) in vitro but translational applications have been hampered by high costs of production and by low rates of enucleation. We have developed R6 and IMIT, two chemically defined culture media and combined them into robust erythroid differentiation (RED) protocols to differentiate induced-pluripotent stem cells (iPSCs) and peripheral blood mononuclear cells (MNCs) into enucleated erythroid cells. The RED protocols do not require any albumin or animal components and require ten- to twentyfold less transferrin (Tf) than previously, because iron is provided to the differentiating erythroblasts by small amounts of recombinant Tf supplemented with FeIII-EDTA, an iron chelator that allows Tf recycling to take place in cell culture. Importantly, cRBCs produced by iPSC differentiation using the long PSC-RED protocol enucleate at much higher rates than with previous protocols, eliminating one of the impediments to the use of these cells to produce clinically useful cRBCs. The absence of albumin, the reduced amounts of Tf, the improved reproducibility associated with the elimination of all animal components, and the high yield on the RED protocols decrease the cost of production of cultured red blood cells. RED protocols should therefore help to make translational applications of cultured RBCs more economically realistic.
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Affiliation(s)
- Emmanuel N Olivier
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Shouping Zhang
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Zi Yan
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Sandra Suzuka
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Karl Roberts
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Kai Wang
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Eric E Bouhassira
- Department of Cell Biology and Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY.
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13
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Bandi S, Tchaikovskaya T, Gupta S. Hepatic differentiation of human pluripotent stem cells by developmental stage-related metabolomics products. Differentiation 2019; 105:54-70. [PMID: 30776728 DOI: 10.1016/j.diff.2019.01.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2018] [Revised: 01/22/2019] [Accepted: 01/28/2019] [Indexed: 12/19/2022]
Abstract
Endogenous cell signals regulate tissue homeostasis and are significant for directing the fate of stem cells. During liver development, cytokines released from various cell types are critical for stem/progenitor cell differentiation and lineage expansions. To determine mechanisms in these stage-specific lineage interactions, we modeled potential effects of soluble signals derived from immortalized human fetal liver parenchymal cells on stem cells, including embryonic and induced pluripotent stem cells. For identifying lineage conversion and maturation, we utilized conventional assays of cell morphology, gene expression analysis and lineage markers. Molecular pathway analysis used functional genomics approaches. Metabolic properties were analyzed to determine the extent of hepatic differentiation. Cell transplantation studies were performed in mice with drug-induced acute liver failure to elicit benefits in hepatic support and tissue regeneration. These studies showed signals emanating from fetal liver cells induced hepatic differentiation in stem cells. Gene expression profiling and comparison of regulatory networks in immature and mature hepatocytes revealed stem cell-derived hepatocytes represented early fetal-like stage. Unexpectedly, differentiation-inducing soluble signals constituted metabolomics products and not proteins. In stem cells exposed to signals from fetal cells, mechanistic gene networks of upstream regulators decreased pluripotency, while simultaneously inducing mesenchymal and epithelial properties. The extent of metabolic and synthetic functions in stem cell-derived hepatocytes was sufficient for providing hepatic support along with promotion of tissue repair to rescue mice in acute liver failure. During this rescue, paracrine factors from transplanted cells contributed in stimulating liver regeneration. We concluded that hepatic differentiation of pluripotent stem cells with metabolomics products will be significant for developing therapies. The differentiation mechanisms involving metabolomics products could have an impact on advancing recruitment of stem/progenitor cells during tissue homeostasis.
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Affiliation(s)
- Sriram Bandi
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
| | - Tatyana Tchaikovskaya
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
| | - Sanjeev Gupta
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Diabetes Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Irwin S. and Sylvia Chanin Institute for Cancer Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
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14
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An HH, Poncz M, Chou ST. Induced Pluripotent Stem Cell-Derived Red Blood Cells, Megakaryocytes, and Platelets: Progress and Challenges. CURRENT STEM CELL REPORTS 2018. [DOI: 10.1007/s40778-018-0144-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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15
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Vanuytsel K, Matte T, Leung A, Naing ZH, Morrison T, Chui DHK, Steinberg MH, Murphy GJ. Induced pluripotent stem cell-based mapping of β-globin expression throughout human erythropoietic development. Blood Adv 2018; 2:1998-2011. [PMID: 30108108 PMCID: PMC6093724 DOI: 10.1182/bloodadvances.2018020560] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2018] [Accepted: 07/09/2018] [Indexed: 02/01/2023] Open
Abstract
Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish β-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with β-globin-expressing cells showing increased maturation. Compared with other cells, β-globin-expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.
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Affiliation(s)
- Kim Vanuytsel
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
- Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, Boston, MA
| | - Taylor Matte
- Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, Boston, MA
| | - Amy Leung
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
- Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, Boston, MA
| | - Zaw Htut Naing
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
- Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, Boston, MA
| | - Tasha Morrison
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
| | - David H K Chui
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
| | - Martin H Steinberg
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
| | - George J Murphy
- Section of Hematology and Medical Oncology, School of Medicine, Boston University, Boston, MA; and
- Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, Boston, MA
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16
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Diepstraten ST, Hart AH. Modelling human haemoglobin switching. Blood Rev 2018; 33:11-23. [PMID: 30616747 DOI: 10.1016/j.blre.2018.06.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 04/11/2018] [Accepted: 06/14/2018] [Indexed: 12/22/2022]
Abstract
Genetic lesions of the β-globin gene result in haemoglobinopathies such as β-thalassemia and sickle cell disease. To discover and test new molecular medicines for β-haemoglobinopathies, cell-based and animal models are now being widely utilised. However, multiple in vitro and in vivo models are required due to the complex structure and regulatory mechanisms of the human globin gene locus, subtle species-specific differences in blood cell development, and the influence of epigenetic factors. Advances in genome sequencing, gene editing, and precision medicine have enabled the first generation of molecular therapies aimed at reactivating, repairing, or replacing silenced or damaged globin genes. Here we compare and contrast current animal and cell-based models, highlighting their complementary strengths, reflecting on how they have informed the scope and direction of the field, and describing some of the novel molecular and precision medicines currently under development or in clinical trial.
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Affiliation(s)
- Sarah T Diepstraten
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC 3086, Australia.
| | - Adam H Hart
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC 3086, Australia.
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17
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Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors. Curr Opin Hematol 2017; 24:283-288. [DOI: 10.1097/moh.0000000000000343] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
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18
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Shen J, Wang ZY, Cheng T. [Generation of engraftable hematopoietic stem cells from human pluripotent stem cells]. ZHONGHUA XUE YE XUE ZA ZHI = ZHONGHUA XUEYEXUE ZAZHI 2017; 38:257-260. [PMID: 28395456 PMCID: PMC7348385 DOI: 10.3760/cma.j.issn.0253-2727.2017.03.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Affiliation(s)
| | | | - T Cheng
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, CAMS & PUMC, Tianjin 300020, China
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19
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Xu Y, Shan W, Li X, Wang B, Liu S, Wang Y, Long Y, Tie R, Wang L, Cai S, Zhang H, Lin Y, Zhang M, Zheng W, Luo Y, Yu X, Yee JK, Ji J, Huang H. A synthetic three-dimensional niche system facilitates generation of functional hematopoietic cells from human-induced pluripotent stem cells. J Hematol Oncol 2016; 9:102. [PMID: 27686241 PMCID: PMC5043527 DOI: 10.1186/s13045-016-0326-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 09/16/2016] [Indexed: 12/18/2022] Open
Abstract
Background The efficient generation of hematopoietic stem cells (HSCs) from human-induced pluripotent stem cells (iPSCs) holds great promise in personalized transplantation therapies. However, the derivation of functional and transplantable HSCs from iPSCs has had very limited success thus far. Methods We developed a synthetic 3D hematopoietic niche system comprising nanofibers seeded with bone marrow (BM)-derived stromal cells and growth factors to induce functional hematopoietic cells from human iPSCs in vitro. Results Approximately 70 % of human CD34+ hematopoietic cells accompanied with CD43+ progenitor cells could be derived from this 3D induction system. Colony-forming-unit (CFU) assay showed that iPSC-derived CD34+ cells formed all types of hematopoietic colonies including CFU-GEMM. TAL-1 and MIXL1, critical transcription factors associated with hematopoietic development, were expressed during the differentiation process. Furthermore, iPSC-derived hematopoietic cells gave rise to both lymphoid and myeloid lineages in the recipient NOD/SCID mice after transplantation. Conclusions Our study underscores the importance of a synthetic 3D niche system for the derivation of transplantable hematopoietic cells from human iPSCs in vitro thereby establishing a foundation towards utilization of human iPSC-derived HSCs for transplantation therapies in the clinic. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0326-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Yulin Xu
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Wei Shan
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Xia Li
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Binsheng Wang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Senquan Liu
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Yebo Wang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Yan Long
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Ruxiu Tie
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Limengmeng Wang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Shuyang Cai
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Hao Zhang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Yu Lin
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Mingming Zhang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Weiyan Zheng
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Yi Luo
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Xiaohong Yu
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - Jiing-Kuan Yee
- Department of Diabetes and Metabolic Diseases Research, City of Hope, Duarte, CA, 91010, USA
| | - Junfeng Ji
- Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, 310012, China
| | - He Huang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310012, China.
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20
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Ackermann M, Liebhaber S, Klusmann JH, Lachmann N. Lost in translation: pluripotent stem cell-derived hematopoiesis. EMBO Mol Med 2016; 7:1388-402. [PMID: 26174486 PMCID: PMC4644373 DOI: 10.15252/emmm.201505301] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage.
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Affiliation(s)
- Mania Ackermann
- RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence Hannover Medical School, Hannover, Germany Institute of Experimental Hematology Hannover Medical School, Hannover, Germany
| | - Steffi Liebhaber
- RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence Hannover Medical School, Hannover, Germany Institute of Experimental Hematology Hannover Medical School, Hannover, Germany
| | | | - Nico Lachmann
- RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence Hannover Medical School, Hannover, Germany Institute of Experimental Hematology Hannover Medical School, Hannover, Germany JRG Translational Hematology of Congenital Diseases, REBIRTH Cluster of Excellence Hannover Medical School, Hannover, Germany
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21
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Dou DR, Calvanese V, Sierra MI, Nguyen AT, Minasian A, Saarikoski P, Sasidharan R, Ramirez CM, Zack JA, Crooks GM, Galic Z, Mikkola HKA. Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nat Cell Biol 2016; 18:595-606. [PMID: 27183470 PMCID: PMC4981340 DOI: 10.1038/ncb3354] [Citation(s) in RCA: 83] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2014] [Accepted: 04/08/2016] [Indexed: 12/18/2022]
Abstract
Pluripotent stem cells (PSC) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had CD34+CD38−/loCD90+CD45+GPI-80+ foetal liver (FL) HSC immunophenotype, but displayed poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, retinoic acid signalling-induced medial HOXA gene expression marks the establishment of the definitive HSC fate and controls HSC identity and function.
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Affiliation(s)
- Diana R Dou
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.,Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Vincenzo Calvanese
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Maria I Sierra
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Andrew T Nguyen
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Arazin Minasian
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Pamela Saarikoski
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Rajkumar Sasidharan
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Christina M Ramirez
- Department of Biostatistics, Fielding School of Public Health, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Jerome A Zack
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.,Department of Medicine, Division of Hematology-Oncology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Gay M Crooks
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.,Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Zoran Galic
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.,Department of Medicine, Division of Hematology-Oncology, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Hanna K A Mikkola
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.,Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.,Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA
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22
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Huang X, Wang Y, Yan W, Smith C, Ye Z, Wang J, Gao Y, Mendelsohn L, Cheng L. Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation. Stem Cells 2016; 33:1470-9. [PMID: 25702619 DOI: 10.1002/stem.1969] [Citation(s) in RCA: 143] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2014] [Accepted: 01/14/2015] [Indexed: 12/11/2022]
Abstract
Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.
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Affiliation(s)
- Xiaosong Huang
- Division of Hematology, Department of Medicine; Institute for Cell Engineering
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23
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Aydin MM, Bayin NS, Acun T, Yakicier MC, Akçali KC. Role of FLT3 in the proliferation and aggressiveness of hepatocellular carcinoma. Turk J Med Sci 2016; 46:572-81. [PMID: 27511526 DOI: 10.3906/sag-1501-173] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2015] [Accepted: 05/26/2015] [Indexed: 11/03/2022] Open
Abstract
BACKGROUND/AIM Previously we showed that Fms-like tyrosine kinase (FLT3) changes its cellular localization upon partial hepatectomy, suggesting a role in liver regeneration. FLT3 was also shown to play an important function in cellular proliferation and activation of PI3K and Ras. Thus, we aimed to investigate the role of FLT3 in hepatocellular tumorigenesis utilizing in vitro and in vivo models. MATERIALS AND METHODS We used Snu398 cells that express FLT3. We investigated these cells' in vitro proliferation and invasion abilities by treatment with the FLT3 inhibitor K-252a or by knocking-down with FLT3 shRNA,. Furthermore, the effect of blocking FLT3 activity and expression during in vivo tumorigenesis was assessed with xenograft models. RESULTS After K-252a treatment or stable knock-down, these cells' proliferation and migration abilities were highly diminished in vitro. In addition, significant diminution in tumorigenicity of Snu398 cells was also obtained in vivo. When FLT3 knocked-down Snu398 cells were injected into nude mice, we did not detect αSMA expression in these tumors, suggesting a role for FLT3 in in vivo invasiveness. CONCLUSION Our data provided evidence that FLT3 has a crucial role both in hepatocarcinogenesis and its invasiveness. Therefore, targeting FLT3 and/or its activity may be a promising tool for combating hepatocellular carcinomas.
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Affiliation(s)
- Muammer Merve Aydin
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Nermin Sumru Bayin
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Tolga Acun
- Department of Molecular Biology and Genetics, Faculty of Sciences and Arts, Bülent Ecevit University, Zonguldak, Turkey
| | | | - Kamil Can Akçali
- Department of Biophysics, Ankara University, Faculty of Medicine, Ankara, Turkey
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24
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Liu S, Xu Y, Zhou Z, Feng B, Huang H. Progress and challenges in generating functional hematopoietic stem/progenitor cells from human pluripotent stem cells. Cytotherapy 2015; 17:344-58. [PMID: 25680303 DOI: 10.1016/j.jcyt.2015.01.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Revised: 01/03/2015] [Accepted: 01/06/2015] [Indexed: 11/25/2022]
Abstract
The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) in vitro holds great potential for providing alternative sources of donor cells for clinical HSC transplantation. However, the low efficiency of current protocols for generating blood lineages and the dysfunction identified in hPSC-derived hematopoietic cells limit their use for full hematopoietic reconstitution in clinics. This review outlines the current understanding of in vitro hematopoietic differentiation from hPSCs, emphasizes the intrinsic and extrinsic molecular mechanisms that are attributed to the aberrant phenotype and function in hPSC-derived hematopoietic cells, pinpoints the current challenges to develop the truly functional HSCs from hPSCs for clinical applications and explores their potential solutions.
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Affiliation(s)
- Senquan Liu
- Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Yulin Xu
- Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
| | - Zijing Zhou
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Bo Feng
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; SBS Core Laboratory, Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
| | - He Huang
- Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
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25
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Kim WS, Zhu Y, Deng Q, Chin CJ, He CB, Grieco AJ, Dravid GG, Parekh C, Hollis RP, Lane TF, Bouhassira EE, Kohn DB, Crooks GM. Erythropoiesis from human embryonic stem cells through erythropoietin-independent AKT signaling. Stem Cells 2015; 32:1503-14. [PMID: 24677652 DOI: 10.1002/stem.1677] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2013] [Revised: 12/16/2013] [Accepted: 01/11/2014] [Indexed: 12/25/2022]
Abstract
Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC.
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Affiliation(s)
- William S Kim
- Department of Pathology and Laboratory Medicine, University of California Los Angeles (UCLA),, Los Angeles, California, USA
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26
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Ramboer E, Vanhaecke T, Rogiers V, Vinken M. Immortalized Human Hepatic Cell Lines for In Vitro Testing and Research Purposes. Methods Mol Biol 2015; 1250:53-76. [PMID: 26272134 PMCID: PMC4579543 DOI: 10.1007/978-1-4939-2074-7_4] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The ubiquitous shortage of primary human hepatocytes has urged the scientific community to search for alternative cell sources, such as immortalized hepatic cell lines. Over the years, several human hepatic cell lines have been produced, whether or not using a combination of viral oncogenes and human telomerase reverse transcriptase protein. Conditional approaches for hepatocyte immortalization have also been established and allow generation of growth-controlled cell lines. A variety of immortalized human hepatocytes have already proven useful as tools for liver-based in vitro testing and fundamental research purposes. The present chapter describes currently applied immortalization strategies and provides an overview of the actually available immortalized human hepatic cell lines and their in vitro applications.
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Affiliation(s)
- Eva Ramboer
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels, 1090, Belgium,
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27
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Dorn I, Klich K, Arauzo-Bravo MJ, Radstaak M, Santourlidis S, Ghanjati F, Radke TF, Psathaki OE, Hargus G, Kramer J, Einhaus M, Kim JB, Kögler G, Wernet P, Schöler HR, Schlenke P, Zaehres H. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin. Haematologica 2014; 100:32-41. [PMID: 25326431 DOI: 10.3324/haematol.2014.108068] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.
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Affiliation(s)
- Isabel Dorn
- Max Planck Institute for Molecular Biomedicine, Münster, Germany Pediatric Hematology and Oncology, University Hospital Münster, Germany
| | - Katharina Klich
- Max Planck Institute for Molecular Biomedicine, Münster, Germany Institute for Transfusion Medicine and Transplantation Immunology, University Hospital Münster, Germany
| | - Marcos J Arauzo-Bravo
- Group of Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute, San Sebastián, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
| | - Martina Radstaak
- Max Planck Institute for Molecular Biomedicine, Münster, Germany
| | - Simeon Santourlidis
- Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, Germany
| | - Foued Ghanjati
- Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, Germany
| | - Teja F Radke
- Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, Germany
| | | | - Gunnar Hargus
- Max Planck Institute for Molecular Biomedicine, Münster, Germany Institute for Neuropathology, University Hospital Münster, Germany
| | - Jan Kramer
- Medical Department I, University of Lübeck, Germany LADR GmbH, Geesthacht, Germany
| | | | - Jeong Beom Kim
- UNIST, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea
| | - Gesine Kögler
- Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, Germany
| | - Peter Wernet
- Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, Germany
| | - Hans R Schöler
- Max Planck Institute for Molecular Biomedicine, Münster, Germany Faculty of Medicine, University of Münster, Germany
| | - Peter Schlenke
- Institute for Transfusion Medicine and Transplantation Immunology, University Hospital Münster, Germany Clinics for Blood Group Serology and Transfusion Medicine, Medical University Graz, Austria
| | - Holm Zaehres
- Max Planck Institute for Molecular Biomedicine, Münster, Germany
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28
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Singh VK, Saini A, Tsuji K, Sharma PB, Chandra R. Manufacturing blood ex vivo: a futuristic approach to deal with the supply and safety concerns. Front Cell Dev Biol 2014; 2:26. [PMID: 25364733 PMCID: PMC4206981 DOI: 10.3389/fcell.2014.00026] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2014] [Accepted: 05/26/2014] [Indexed: 12/13/2022] Open
Abstract
Blood transfusions are routinely done in every medical regimen and a worldwide established collection, processing/storage centers provide their services for the same. There have been extreme global demands for both raising the current collections and supply of safe/adequate blood due to increasingly demanding population. With, various risks remain associated with the donor derived blood, and a number of post collection blood screening and processing methods put extreme constraints on supply system especially in the underdeveloped countries. A logistic approach to manufacture erythrocytes ex-vivo by using modern tissue culture techniques have surfaced in the past few years. There are several reports showing the possibilities of RBCs (and even platelets/neutrophils) expansion under tightly regulated conditions. In fact, ex vivo synthesis of the few units of clinical grade RBCs from a single dose of starting material such as umbilical cord blood (CB) has been well established. Similarly, many different sources are also being explored for the same purpose, such as embryonic stem cells, induced pluripotent stem cells. However, the major concerns remain elusive before the manufacture and clinical use of different blood components may be used to successfully replace the present system of donor derived blood transfusion. The most important factor shall include the large scale of RBCs production from each donated unit within a limited time period and cost of their production, both of these issues need to be handled carefully since many of the recipients among developing countries are unable to pay even for the freely available donor derived blood. Anyways, keeping these issues in mind, present article shall be focused on the possibilities of blood production and their use in the near future.
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Affiliation(s)
- Vimal K Singh
- Stem Cell Research Laboratory, Department of Biotechnology, Delhi Technological University Delhi, India
| | - Abhishek Saini
- Stem Cell Research Laboratory, Department of Biotechnology, Delhi Technological University Delhi, India
| | - Kohichiro Tsuji
- Departments of Pediatric Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo Hospital Tokyo, Japan
| | - P B Sharma
- Stem Cell Research Laboratory, Department of Biotechnology, Delhi Technological University Delhi, India
| | - Ramesh Chandra
- Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi Delhi, India
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29
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Baron MH. Concise Review: early embryonic erythropoiesis: not so primitive after all. Stem Cells 2014; 31:849-56. [PMID: 23361843 DOI: 10.1002/stem.1342] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2012] [Accepted: 12/27/2012] [Indexed: 12/28/2022]
Abstract
In the developing embryo, hematopoiesis begins with the formation of primitive erythroid cells (EryP), a distinct and transient red blood cell lineage. EryP play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Progenitors for EryP arise as a cohort within the blood islands of the mammalian yolk sac at the end of gastrulation. As a strong heartbeat is established, nucleated erythroblasts begin to circulate and to mature in a stepwise, nearly synchronous manner. Until relatively recently, these cells were thought to be "primitive" in that they seemed to more closely resemble the nucleated erythroid cells of lower vertebrates than the enucleated erythrocytes of mammals. It is now known that mammalian EryP do enucleate, but not until several days after entering the bloodstream. I will summarize the common and distinguishing characteristics of primitive versus definitive (adult-type) erythroid cells, review the development of EryP from the emergence of their progenitors through maturation and enucleation, and discuss pluripotent stem cells as models for erythropoiesis. Erythroid differentiation of both mouse and human pluripotent stem cells in vitro has thus far reproduced early but not late red blood cell ontogeny. Therefore, a deeper understanding of cellular and molecular mechanisms underlying the differences and similarities between the embryonic and adult erythroid lineages will be critical to improving methods for production of red blood cells for use in the clinic.
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Affiliation(s)
- Margaret H Baron
- Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
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30
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Nakajima-Takagi Y, Osawa M, Iwama A. Manipulation of Hematopoietic Stem Cells for Regenerative Medicine. Anat Rec (Hoboken) 2013; 297:111-20. [DOI: 10.1002/ar.22804] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Accepted: 09/13/2013] [Indexed: 12/25/2022]
Affiliation(s)
- Yaeko Nakajima-Takagi
- Department of Cellular and Molecular Medicine; Graduate School of Medicine; Chiba University; 1-8-1 Inohana Chuo-ku Chiba 260-8670 Japan
- Japan Science and Technology Corporation, Core Research for Evolutional Science and Technology; Gobancho Chiyoda-ku, Tokyo Japan
| | - Mitsujiro Osawa
- Department of Cellular and Molecular Medicine; Graduate School of Medicine; Chiba University; 1-8-1 Inohana Chuo-ku Chiba 260-8670 Japan
- Japan Science and Technology Corporation, Core Research for Evolutional Science and Technology; Gobancho Chiyoda-ku, Tokyo Japan
| | - Atsushi Iwama
- Department of Cellular and Molecular Medicine; Graduate School of Medicine; Chiba University; 1-8-1 Inohana Chuo-ku Chiba 260-8670 Japan
- Japan Science and Technology Corporation, Core Research for Evolutional Science and Technology; Gobancho Chiyoda-ku, Tokyo Japan
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31
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Mazurier C, Douay L. [Red blood cell production for transfusion purposes. A stem cell ex vivo fate]. Transfus Clin Biol 2013; 20:90-4. [PMID: 23601197 DOI: 10.1016/j.tracli.2013.02.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
In vitro generation of red blood cells (RBC) makes sense in a context of recurrent shortage. It could be an interesting complementary source for "classic" transfusion products by combining the sufficiency of supply, homemade production of a particular phenotype of interest and reduced risk of infection. The question that arises is how to produce in vitro RBC? Here we retrace the steps that led to the production of functional RBC, from basic knowledge of in vivo erythropoiesis to in vitro generation of RBC from different sources of stem cells. Regarding the adults HSC, the major finding lies in the recent establishment of proof of concept of their transfusion in humans. Because the induced pluripotent stem cells (IPS) can proliferate indefinitely and be selected for a phenotype of interest, they are obviously the best source of stem cells. Proof of concept of generation of RBC from IPS has been made, but still has to be optimized. We also discuss the key points that need to be solved to achieve clinical transfusion application.
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Affiliation(s)
- C Mazurier
- Établissement français du sang Île-de-France, 94200 Ivry-sur-Seine, France
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32
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Bouhassira EE. Concise review: production of cultured red blood cells from stem cells. Stem Cells Transl Med 2012; 1:927-33. [PMID: 23283554 PMCID: PMC3659674 DOI: 10.5966/sctm.2012-0097] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Accepted: 10/04/2012] [Indexed: 01/11/2023] Open
Abstract
In the Western world, the volunteer-based collection system covers most transfusion needs, but transient shortages regularly develop and blood supplies are vulnerable to potentially major disruptions. The production of cultured red blood cells from stem cells is slowly emerging as a potential alternative. The various cell sources, the niche applications most likely to reach the clinic first, and some of the remaining technical issues are reviewed here.
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Affiliation(s)
- Eric E Bouhassira
- Departments of Cell Biology and Medicine, Albert Einstein College of Medicine, New York, NY, USA.
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33
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Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition. Blood 2012; 121:770-80. [PMID: 23169780 DOI: 10.1182/blood-2012-07-444208] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
UNLABELLED Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.
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34
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Olivier E, Qiu C, Bouhassira EE. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood. Stem Cells Transl Med 2012. [PMID: 23197866 DOI: 10.5966/sctm.2012-0059] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.
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Affiliation(s)
- Emmanuel Olivier
- Departments of Medicine and Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA
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35
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Ebihara Y, Ma F, Tsuji K. Generation of red blood cells from human embryonic/induced pluripotent stem cells for blood transfusion. Int J Hematol 2012; 95:610-6. [PMID: 22648827 DOI: 10.1007/s12185-012-1107-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2012] [Revised: 05/14/2012] [Accepted: 05/14/2012] [Indexed: 12/19/2022]
Abstract
Red blood cell (RBC) transfusion is necessary for many patients with emergency or hematological disorders. However, to date the supply of RBCs remains labile and dependent on voluntary donations. In addition, the transmission of infectious disease via blood transfusion from unspecified donors remains a risk. Establishing a large quantity of safe RBCs would help to address this issue. Human embryonic stem (hES) cells and the recently established human induced pluripotent stem (hiPS) cells represent potentially unlimited sources of donor-free RBCs for blood transfusion, as they can proliferate indefinitely in vitro. Extensive research has been done to efficiently generate transfusable RBCs from hES/iPS cells. Nevertheless, a number of challenges must be overcome before the clinical usage of hES/iPS cell-derived RBCs can become a reality.
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Affiliation(s)
- Yasuhiro Ebihara
- Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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36
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Diaz-Solano D, Wittig O, Ayala-Grosso C, Pieruzzini R, Cardier JE. Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells. Stem Cells Dev 2012; 21:3187-96. [PMID: 22471939 DOI: 10.1089/scd.2012.0084] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.
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Affiliation(s)
- Dylana Diaz-Solano
- Unidad de Terapia Celular-Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela
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37
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Modeling human hematopoietic cell development from pluripotent stem cells. Exp Hematol 2012; 40:601-11. [PMID: 22510344 DOI: 10.1016/j.exphem.2012.04.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2012] [Accepted: 04/04/2012] [Indexed: 11/20/2022]
Abstract
Understanding the steps and cues that allow hematopoietic cells to be generated during development holds great clinical as well as biological interest. Analysis of these events in mice has provided many important insights into the processes involved, but features that might be unique to humans remain challenging to elucidate because they cannot be studied directly in vivo. Human embryonic stem or induced pluripotent stem cells offer attractive in vitro alternatives to analyze the process. Here we review recent efforts to develop defined and quantitative systems to address outstanding developmental questions against a background of what we know about the development of hematopoietic cells in the fetus and derived from mouse embryonic stem cells.
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38
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Xu Y, Liu L, Zhang L, Fu S, Hu Y, Wang Y, Fu H, Wu K, Xiao H, Liu S, Yu X, Zheng W, Feng B, Huang H. Efficient commitment to functional CD34+ progenitor cells from human bone marrow mesenchymal stem-cell-derived induced pluripotent stem cells. PLoS One 2012; 7:e34321. [PMID: 22496789 PMCID: PMC3322134 DOI: 10.1371/journal.pone.0034321] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2011] [Accepted: 02/28/2012] [Indexed: 01/14/2023] Open
Abstract
The efficient commitment of a specialized cell type from induced pluripotent stem cells (iPSCs) without contamination from unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone marrow mesenchymal stem cells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal, hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P<0.05). After culturing with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7–9 days, CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or from the spontaneous differentiation groups (P<0.05), as assessed by flow cytometry analysis. These induced cells expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of the mechanisms of hematopoiesis and drug screening.
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Affiliation(s)
- Yulin Xu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Lizhen Liu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Lifei Zhang
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Shan Fu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Yongxian Hu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Yingjia Wang
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Huarui Fu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Kangni Wu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Haowen Xiao
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Senquan Liu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Xiaohong Yu
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Weiyan Zheng
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
| | - Bo Feng
- School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China
| | - He Huang
- Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
- * E-mail:
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39
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Chen Q, Fabry ME, Rybicki AC, Suzuka SM, Balazs TC, Etzion Z, de Jong K, Akoto EK, Canterino JE, Kaul DK, Kuypers FA, Lefer D, Bouhassira EE, Hirsch RE. A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress. Blood Cells Mol Dis 2012; 48:91-101. [PMID: 22260787 PMCID: PMC3310900 DOI: 10.1016/j.bcmd.2011.12.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2011] [Revised: 11/04/2011] [Accepted: 11/22/2011] [Indexed: 11/19/2022]
Abstract
Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid β(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of β(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.
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Affiliation(s)
- Qiuying Chen
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Mary E. Fabry
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Anne C. Rybicki
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
- Montefiore Medical Center, Bronx, NY
| | - Sandra M. Suzuka
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Tatiana C. Balazs
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Zipora Etzion
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Kitty de Jong
- Children’s Hospital of Oakland, Research Institute, CA
| | - Edna K. Akoto
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Joseph E. Canterino
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | - Dhananjay K. Kaul
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
| | | | - David Lefer
- Department of Surgery, Emory University School of Medicine, Atlanta, Ga
| | - Eric E. Bouhassira
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY
| | - Rhoda Elison Hirsch
- Department of Medicine/Hematology, Albert Einstein College of Medicine, Bronx, NY
- Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, NY
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40
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Chang KH, Bonig H, Papayannopoulou T. Generation and characterization of erythroid cells from human embryonic stem cells and induced pluripotent stem cells: an overview. Stem Cells Int 2011; 2011:791604. [PMID: 22135684 PMCID: PMC3205655 DOI: 10.4061/2011/791604] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Accepted: 08/29/2011] [Indexed: 12/29/2022] Open
Abstract
Because of the imbalance in the supply and demand of red blood cells (RBCs), especially for alloimmunized patients or patients with rare blood phenotypes, extensive research has been done to generate therapeutic quantities of mature RBCs from hematopoietic stem cells of various sources, such as bone marrow, peripheral blood, and cord blood. Since human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be maintained indefinitely in vitro, they represent potentially inexhaustible sources of donor-free RBCs. In contrast to other ex vivo stem-cell-derived cellular therapeutics, tumorigenesis is not a concern, as RBCs can be irradiated without marked adverse effects on in vivo function. Here, we provide a comprehensive review of the recent publications relevant to the generation and characterization of hESC- and iPSC-derived erythroid cells and discuss challenges to be met before the eventual realization of clinical usage of these cells.
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Affiliation(s)
- Kai-Hsin Chang
- Division of Hematology, Department of Medicine, University of Washington, 1705 NE Pacific, Rm K243, P. O. Box 357710, Seattle, WA 98195-7710, USA
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Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells. PLoS One 2011; 6:e25761. [PMID: 22022444 PMCID: PMC3192723 DOI: 10.1371/journal.pone.0025761] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2011] [Accepted: 09/12/2011] [Indexed: 12/20/2022] Open
Abstract
We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.
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Pourcher G, Mazurier C, King YY, Giarratana MC, Kobari L, Boehm D, Douay L, Lapillonne H. Human fetal liver: an in vitro model of erythropoiesis. Stem Cells Int 2011; 2011:405429. [PMID: 21961016 PMCID: PMC3179878 DOI: 10.4061/2011/405429] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2011] [Accepted: 05/24/2011] [Indexed: 01/15/2023] Open
Abstract
We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34(+) cells. In this in vitro model, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramatic in vitro expansion (100-fold more when compared to CB CD34(+)) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10-15% cloning efficiency for adult CD34(+) cells. This work supports the idea that FL remains a model of study and is not a candidate for ex vivo RBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.
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Affiliation(s)
- Guillaume Pourcher
- Prolifération et Différenciation des Cellules Souches: Application à la Thérapie Cellulaire Hématopoïétique, INSERM, UMR_S938, CDR Saint-Antoine, 75012 Paris, France
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Peyrard T, Bardiaux L, Krause C, Kobari L, Lapillonne H, Andreu G, Douay L. Banking of pluripotent adult stem cells as an unlimited source for red blood cell production: potential applications for alloimmunized patients and rare blood challenges. Transfus Med Rev 2011; 25:206-16. [PMID: 21377319 DOI: 10.1016/j.tmrv.2011.01.002] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The transfusion of red blood cells (RBCs) is now considered a well-settled and essential therapy. However, some difficulties and constraints still occur, such as long-term blood product shortage, blood donor population aging, known and yet unknown transfusion-transmitted infectious agents, growing cost of the transfusion supply chain management, and the inescapable blood group polymorphism barrier. Red blood cells can be now cultured in vitro from human hematopoietic, human embryonic, or human-induced pluripotent stem cells (hiPSCs). The highly promising hiPSC technology represents a potentially unlimited source of RBCs and opens the door to the revolutionary development of a new generation of allogeneic transfusion products. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, we draw here some futuristic but realistic scenarios regarding potential applications for alloimmunized patients and those with a rare blood group. We retrospectively studied a cohort of 16,486 consecutive alloimmunized patients (10-year period), showing 1 to 7 alloantibodies with 361 different antibody combinations. We showed that only 3 hiPSC clones would be sufficient to match more than 99% of the 16,486 patients in need of RBC transfusions. The study of the French National Registry of People with a Rare Blood Phenotype/Genotype (10-year period) shows that 15 hiPSC clones would cover 100% of the needs in patients of white ancestry. In addition, one single hiPSC clone would meet 73% of the needs in alloimmunized patients with sickle cell disease for whom rare cryopreserved RBC units were required. As a result, we consider that a very limited number of RBC clones would be able to not only provide for the need for most alloimmunized patients and those with a rare blood group but also efficiently allow for a policy for alloimmunization prevention in multiply transfused patients.
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Abstract
Blood transfusion is a mainstay of modern clinical medicine. However, a number of fundamental problems persist, including insufficiency of supply, the threat of transfusion transmissible infectious disease and the problem of immune incompatibility. It would be extremely valuable, therefore, to develop a potentially limitless, infection free, immune neutral source of erythrocytes for transfusion. Human embryonic stem cells (hESC), have potentially limitless proliferative capacity and the potential to differentiate into the majority of adult cell types including erythrocytes. A number of barriers to the development of clinical cellular therapeutics from hESC have been posited, including HLA incompatibility between donor and recipient, difficulties in defining optimal cell phenotype and function in vitro and the fact that most tissues consist of complex three-dimensional matrices of cells. Many or most of these problems are circumvented in the generation of erythrocytes and group O RhD negative Kell negative blood would be compatible with the majority of recipients. Red cell transfusion is therefore an attractive goal for pluripotent stem cell derived therapeutics. Much progress has been made however, a number of challenges remain including scale up, ensuring clinical effectiveness and product safety.
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Affiliation(s)
- Joanne C Mountford
- Scottish National Blood Transfusion Service, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
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Slukvin II. Renin-angiotensin system and hemangioblast development from human embryonic stem cells. Expert Rev Hematol 2011; 2:137-43. [PMID: 21083448 DOI: 10.1586/ehm.09.4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Human embryonic stem cells (hESCs) offer the opportunity to create a novel source of blood cells for transfusion, transplantation and cancer immunotherapy. Identification of sequential progenitors leading to blood development, as well as a detailed understanding of the molecular mechanisms of hematopoietic lineage specification and diversification from hESCs, will be critical to advance technologies for large-scale production of blood cells and in vitro generation of hematopoietic stem cells. Multiple lines of evidence suggest that hematopoiesis, both in vivo during embryogenesis and in vitro from hESCs, is initiated from hemangioblasts; cells with the potential to generate both hematopoietic and endothelial cells. However, the phenotypic and functional properties of hemangioblasts remain largely unknown. The paper from Zambidis et al. is the first demonstration that hemangioblasts generated from hESCs express angiotensin-converting enzyme (CD143). More importantly, the current study demonstrates that the renin-angiotensin system plays a critical role in the hemangioblast fate decision to produce either blood or endothelial cells. These findings could be exploited for developing novel cellular and drug therapies for hematological and vascular diseases.
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Affiliation(s)
- Igor I Slukvin
- Department of Pathology and Laboratory Medicine, Wisconsin National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA.
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Choi KD, Vodyanik M, Slukvin II. Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc 2011; 6:296-313. [PMID: 21372811 PMCID: PMC3066067 DOI: 10.1038/nprot.2010.184] [Citation(s) in RCA: 121] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.
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Affiliation(s)
- Kyung-Dal Choi
- National Primate Research Center, University of Wisconsin Graduate School, 1220 Capitol Court, Madison, WI 53715
| | - Maxim Vodyanik
- National Primate Research Center, University of Wisconsin Graduate School, 1220 Capitol Court, Madison, WI 53715
| | - Igor I. Slukvin
- National Primate Research Center, University of Wisconsin Graduate School, 1220 Capitol Court, Madison, WI 53715
- Department of Pathology and Laboratory Medicine, University of Wisconsin, 600 Highland Ave., Madison, WI 53792
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Liu D, He X, Wang K, He C, Shi H, Jian L. Biocompatible silica nanoparticles-insulin conjugates for mesenchymal stem cell adipogenic differentiation. Bioconjug Chem 2010; 21:1673-84. [PMID: 20735012 DOI: 10.1021/bc100177v] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
There is increasing interest in developing bioconjugated carriers for the cellular delivery of bioactive molecules to stem cells, since they can allow modulation of stem cell differentiation. The present study reported biocompatible silica nanoparticle-insulin conjugates for rat mesenchymal stem cell (RMSC) adipogenic differentiation in vitro. A systematic study was first carried out on the biocompatibility of the SiNPs with RMSCs. The cell viability assay was performed to screen the SiNP concentration for creating little cytotoxicity on RMSCs. Furthermore, transmission electron microscopy (TEM) and adipogenesis and osteogenesis assays revealed that the pure SiNPs had no effect on cellular ultrastructures, adipogenic differentiation, and osteogenic differentiation. Under the optimized SiNP concentration with little cytotoxicity on RMSC and no effects on the RMSC phenotype, SiNP-insulin conjugates were prepared and used for RMSC adipogenic differentiation. Results showed that RMSCs had the ability to differentiate into adipocytes when cultured in the presence of insulin-conjugated SiNPs. This work demonstrated that the biological activity of insulin conjugated to the SiNPs was not affected and the SiNPs could be used as biocompatibile carriers of insulin for RMSC adipogenic differentiation, which would help to expand the new potential application of SiNPs in stem cell research.
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Affiliation(s)
- Dan Liu
- Institute of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, No. 1 Lushan South Road, Changsha, China
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Ye Z, Cheng L. Potential of human induced pluripotent stem cells derived from blood and other postnatal cell types. Regen Med 2010; 5:521-30. [PMID: 20632856 DOI: 10.2217/rme.10.38] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Human induced pluripotent stem (iPS) cells have been generated from various cell types including blood cells, and offer certain advantages as a starting population for reprogramming postnatal somatic cells. Unlike adult stem cells, iPS cells can proliferate limitlessly in culture while retaining their potential to differentiate into any cell type, including hematopoietic lineages. Derivation of patient-specific iPS cells, in combination with improved hematopoietic differentiation protocols, provides an alternative to generate histocompatible stem cells for bone marrow transplantation. In addition, the ability to reprogram blood cells and redifferentiate iPS cells back to hematopoietic lineages provides opportunities to establish novel models for acquired and inherited blood diseases. This article will summarize recent progress in human iPS cells derived from blood cells and hematopoietic differentiation from iPS cells. Advantages of blood as a source for reprogramming and applications in regenerative medicine will be discussed.
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Affiliation(s)
- Zhaohui Ye
- Division of Hematology & Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Broadway Research Building, Room 747, 733 N. Broadway, Baltimore, MD 21205, USA
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Nodal/Activin signaling predicts human pluripotent stem cell lines prone to differentiate toward the hematopoietic lineage. Mol Ther 2010; 18:2173-81. [PMID: 20736931 DOI: 10.1038/mt.2010.179] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification.
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Liu YX, Yue W, Ji L, Nan X, Pei XT. Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment. BMC DEVELOPMENTAL BIOLOGY 2010; 10:85. [PMID: 20696076 PMCID: PMC2929223 DOI: 10.1186/1471-213x-10-85] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/01/2010] [Accepted: 08/10/2010] [Indexed: 11/10/2022]
Abstract
BACKGROUND We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays. RESULTS The erythroid cells produced from hEBs could differentiate into enucleated cells and expressed globins in a time-dependent manner. They expressed not only embryonic globins but also the adult-globin with the maturation of the erythroid cells. In addition, our data showed that the hEBs-derived erythroid cells were able to act as oxygen carriers, indicating that hESCs could generate functional mature erythroid cells. CONCLUSION Cell extract exposure with the addition of cytokines resulted in robust erythroid -like differentiation of hEBs and these hEBs-derived erythroid cells possessed functions similar to mature red blood cells.
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Affiliation(s)
- Yu-xiao Liu
- Stem Cell and Regenerative Medicine Lab, Beijing Institution of Transfusion Medicine, Beijing 100850, China
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