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1
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Park SA, Shin A, Im E, Yu DH, Kim M, Kim CG, Baek EJ. Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells. Anim Cells Syst (Seoul) 2025; 29:251-263. [PMID: 40304014 PMCID: PMC12039424 DOI: 10.1080/19768354.2025.2492148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2025] [Accepted: 03/11/2025] [Indexed: 05/02/2025] Open
Abstract
Blood transfusion is a critical, lifesaving medical procedure for dogs. However, the limited availability of blood donors and ethical concerns highlight the need for alternative solutions, such as in vitro-produced red blood cells (RBCs), which remain unexplored in canines. This study aimed to produce canine erythrocytes in vitro from peripheral blood (PB) mononuclear cells (MNCs), optimize culture conditions using either human or canine reagents, and identify relevant cell markers. Results indicated that canine erythropoiesis can be induced by human or canine cytokines, producing RBCs within approximately 20 days. Although cell numbers decreased during the first seven days, immature erythroid cells proliferated, reaching peak expansion and RBC production by day 17. Despite the smaller cell size of the cultured RBCs than that of humans, the morphology at each stage of erythroid maturation was analogous to that of human erythropoiesis. Furthermore, the expression patterns of canine alpha hemoglobin stabilizing protein and erythropoietin receptor mirrored those observed in human erythropoiesis. Oxygen-hemoglobin (oxygen-Hb) association and dissociation curves of cultured RBCs closely resembled those of native canine RBCs, indicating an appropriate oxygen-carrying capacity. This study presents the first evidence of successful in vitro production of canine RBCs, offering a promising tool for research and potential therapeutic applications.
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Affiliation(s)
- Sung Ah Park
- Department of Translational Medicine, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea
| | - Arim Shin
- Department of Translational Medicine, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea
| | - Eunji Im
- Department of Research and Development, ArtBlood Inc., Seoul, Korea
| | - Do Hyeon Yu
- College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea
| | - Minji Kim
- College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea
| | - Chul Geun Kim
- Department of Life Science, Hanyang University, Seoul, Korea
| | - Eun Jung Baek
- Department of Translational Medicine, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea
- Department of Research and Development, ArtBlood Inc., Seoul, Korea
- Department of Laboratory Medicine, College of Medicine, Hanyang University, Seoul, Korea
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2
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Claessen MJAG, Yagci N, van Mierlo G, Kersten MJ, von Lindern M, van den Akker E. Human serum albumin or polyvinyl alcohol can only partially replace human plasma during in vitro red cell production from PBMC. Sci Rep 2025; 15:12058. [PMID: 40199875 PMCID: PMC11978868 DOI: 10.1038/s41598-024-81341-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2024] [Accepted: 11/26/2024] [Indexed: 04/10/2025] Open
Abstract
Transfusion of donor-derived red blood cells (RBC) depends on donor availability. Alloimmunization can limit the availability of transfusion units, particularly for chronically transfused patients. In vitro cultured, customizable RBC (cRBC) would negate these concerns and provide infinite RBC products. Previously, we developed a defined medium based on good manufacturing practice (GMP) requirements. To optimize medium conditions with regards to reproducibility and cost effectiveness, we tested the requirement for plasma during the differentiation phase and the replacement of HSA by polyvinyl alcohol (PVA) during the expansion and differentiation phase. We show that 5% plasma is essential to enhance cell count, enucleation% and mostly stability of cRBC during the differentiation phase. During the expansion phase HSA could be replaced by PVA without compromising the expansion capacity. Substitution of HSA by PVA even increased the number of cells at the end of the expansion phase. During the differentiation phase PVA could also replace HSA, but only in the presence of plasma. Plasma is still essential to achieve an optimum yield of enucleated cRBC, likely by stabilizing enucleated cRBC. Substitution of HSA by PVA is a new advancement in the development of a, defined, cost-effective culture medium to culture cRBC for all.
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Affiliation(s)
- Marie-José A G Claessen
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
| | - Nurcan Yagci
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - Gerard van Mierlo
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - Marie José Kersten
- Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
| | - Marieke von Lindern
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - Emile van den Akker
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
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3
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Shah AP, Majeti KR, Ekman FK, Selvaraj S, Sharma D, Sinha R, Soupene E, Chati P, Luna SE, Charlesworth CT, McCreary T, Lesch BJ, Tran T, Chu SN, Porteus MH, Kyle Cromer M. Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis. Nat Commun 2025; 16:1140. [PMID: 39880867 PMCID: PMC11779867 DOI: 10.1038/s41467-025-56239-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2024] [Accepted: 01/10/2025] [Indexed: 01/31/2025] Open
Abstract
Blood transfusion plays a vital role in modern medicine, but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution, yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development, and EPO is among the most expensive media components. To address this challenge, we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells, we achieve equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors, potentially improving scalable manufacturing of a wide range of clinically relevant cell types.
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Affiliation(s)
- Aadit P Shah
- School of Medicine, Stanford University, Stanford, CA, USA
- Department of Pediatrics, Stanford University, Stanford, CA, USA
| | - Kiran R Majeti
- Department of Pediatrics, Stanford University, Stanford, CA, USA
| | - Freja K Ekman
- School of Medicine, Stanford University, Stanford, CA, USA
- Department of Pediatrics, Stanford University, Stanford, CA, USA
- Department of Genetics, Stanford University, Stanford, CA, USA
| | - Sridhar Selvaraj
- Department of Pediatrics, Stanford University, Stanford, CA, USA
| | - Devesh Sharma
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Roshani Sinha
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Eric Soupene
- Benioff Children's Hospital Oakland, University of California, San Francisco, San Francisco, CA, USA
| | - Prathamesh Chati
- Department of Biological & Medical Informatics, University of California, San Francisco, San Francisco, CA, USA
| | - Sofia E Luna
- School of Medicine, Stanford University, Stanford, CA, USA
- Department of Pediatrics, Stanford University, Stanford, CA, USA
| | | | - Travis McCreary
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Benjamin J Lesch
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Tammy Tran
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Simon N Chu
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA
| | | | - M Kyle Cromer
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA.
- Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, USA.
- Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.
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4
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Yan H, Zang R, Cui T, Liu Y, Zhang B, Zhao L, Li H, Zhou J, Wang H, Zeng Q, Xu L, Zhou Y, Pei X, Xi J, Yue W. PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells. Stem Cell Res Ther 2024; 15:310. [PMID: 39294765 PMCID: PMC11412063 DOI: 10.1186/s13287-024-03910-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2024] [Accepted: 08/28/2024] [Indexed: 09/21/2024] Open
Abstract
BACKGROUND Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. METHODS In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells. RESULTS Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. CONCLUSIONS VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.
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Affiliation(s)
- Hao Yan
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Ruge Zang
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China.
| | - Tiantian Cui
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Yiming Liu
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Biao Zhang
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Lingpin Zhao
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Hongyu Li
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Juannian Zhou
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Haiyang Wang
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Quan Zeng
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Lei Xu
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Yuqi Zhou
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Xuetao Pei
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
| | - Jiafei Xi
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China.
| | - Wen Yue
- Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China.
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5
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Claessen MJAG, Yagci N, Fu K, Brandsma E, Kersten MJ, von Lindern M, van den Akker E. Production and stability of cultured red blood cells depends on the concentration of cholesterol in culture medium. Sci Rep 2024; 14:15592. [PMID: 38971841 PMCID: PMC11227516 DOI: 10.1038/s41598-024-66440-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2024] [Accepted: 07/01/2024] [Indexed: 07/08/2024] Open
Abstract
The production of cultured red blood cells (cRBC) for transfusion purposes requires large scale cultures and downstream processes to purify enucleated cRBC. The membrane composition, and cholesterol content in particular, are important during proliferation of (pro)erythroblasts and for cRBC quality. Therefore, we tested the requirement for cholesterol in the culture medium during expansion and differentiation of erythroid cultures with respect to proliferation, enucleation and purification by filtration. The low cholesterol level (22 µg/dl) in serum free medium was sufficient to expand (pro)erythroblast cultures. Addition of 2.0 or 5.0 mg/dL of free cholesterol at the start of differentiation induction inhibited enucleation compared to the default condition containing 3.3 mg/dl total cholesterol derived from the addition of Omniplasma to serum free medium. Addition of 5.0 mg/dl cholesterol at day 5 of differentiation did not affect the enucleation process but significantly increased recovery of enucleated cRBC following filtration over leukodepletion filters. The addition of cholesterol at day 5 increased the osmotic resistance of cRBC. In conclusion, cholesterol supplementation after the onset of enucleation improved the robustness of cRBC and increased the yield of enucleated cRBC in the purification process.
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Affiliation(s)
- M J A G Claessen
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - N Yagci
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - K Fu
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - E Brandsma
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Department of Life Sciences, Saxion University of Applied Sciences, M.H. Tromplaan 28, 7513AB, Enschede, The Netherlands
| | - M J Kersten
- Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands
| | - M von Lindern
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands
| | - E van den Akker
- Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
- Landsteiner Laboratory, Amsterdam University Medical Center, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
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6
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Wang E, Liu S, Zhang X, Peng Q, Yu H, Gao L, Xie A, Ma D, Zhao G, Cheng L. An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2303471. [PMID: 38481061 PMCID: PMC11165465 DOI: 10.1002/advs.202303471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 11/01/2023] [Indexed: 06/12/2024]
Abstract
The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL-carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically-defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.
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Affiliation(s)
- Enyu Wang
- Department of HematologyThe First Affiliated Hospital of USTCDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230001China
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
- Department of Electronic Engineering and Information ScienceUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Senquan Liu
- Department of HematologyThe First Affiliated Hospital of USTCDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230001China
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Xinye Zhang
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Qingyou Peng
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Huijuan Yu
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Lei Gao
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - An Xie
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Ding Ma
- Department of HematologyThe First Affiliated Hospital of USTCDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230001China
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Gang Zhao
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
- Department of Electronic Engineering and Information ScienceUniversity of Science and Technology of ChinaHefeiAnhui230027China
| | - Linzhao Cheng
- Department of HematologyThe First Affiliated Hospital of USTCDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230001China
- Blood and Cell Therapy InstituteAnhui Provincial Key Laboratory of Blood Research and ApplicationsUniversity of Science and Technology of ChinaHefeiAnhui230027China
- School of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefeiAnhui230027China
- Division of HematologyJohns Hopkins University School of MedicineBaltimoreMD21205USA
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7
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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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8
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Jiang JH, Ren RT, Cheng YJ, Li XX, Zhang GR. Immune cells and RBCs derived from human induced pluripotent stem cells: method, progress, prospective challenges. Front Cell Dev Biol 2024; 11:1327466. [PMID: 38250324 PMCID: PMC10796611 DOI: 10.3389/fcell.2023.1327466] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Accepted: 12/14/2023] [Indexed: 01/23/2024] Open
Abstract
Blood has an important role in the healthcare system, particularly in blood transfusions and immunotherapy. However, the occurrence of outbreaks of infectious diseases worldwide and seasonal fluctuations, blood shortages are becoming a major challenge. Moreover, the narrow specificity of immune cells hinders the widespread application of immune cell therapy. To address this issue, researchers are actively developing strategies for differentiating induced pluripotent stem cells (iPSCs) into blood cells in vitro. The establishment of iPSCs from terminally differentiated cells such as fibroblasts and blood cells is a straightforward process. However, there is need for further refinement of the protocols for differentiating iPSCs into immune cells and red blood cells to ensure their clinical applicability. This review aims to provide a comprehensive overview of the strategies and challenges facing the generation of iPSC-derived immune cells and red blood cells.
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Affiliation(s)
- Jin-he Jiang
- Shandong Yinfeng Academy of Life Science, Jinan, Shandong, China
| | - Ru-tong Ren
- Shandong Yinfeng Academy of Life Science, Jinan, Shandong, China
| | - Yan-jie Cheng
- Institute of Biomedical and Health Science, School of Life and Health Science, Anhui Science and Technology University, Chuzhou, Anhui, China
| | - Xin-xin Li
- Shandong Yinfeng Academy of Life Science, Jinan, Shandong, China
| | - Gui-rong Zhang
- Shandong Yinfeng Academy of Life Science, Jinan, Shandong, China
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9
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Boccacci Y, Dumont N, Doyon Y, Laganière J. Accessory-cell-free differentiation of hematopoietic stem and progenitor cells into mature red blood cells. Cytotherapy 2023; 25:1242-1248. [PMID: 37598334 DOI: 10.1016/j.jcyt.2023.07.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 07/14/2023] [Accepted: 07/25/2023] [Indexed: 08/21/2023]
Abstract
BACKGROUND AIMS The culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis, i.e., the terminal maturation of reticulocytes into erythrocytes, so that it can occur without feeder or accessory cells and animal-derived components. METHODS Here, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and xeno-free conditions. RESULTS The approach improves post-enucleation cell integrity and cell survival, and it enables subsequent storage of cRBCs for up to 42 days in classical additive solution conditions without any specialized equipment. CONCLUSIONS We foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs.
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Affiliation(s)
- Yelena Boccacci
- Medical Affairs and Innovation, Héma-Qubec, Québec, Quebec, Canada; Centre Hospitalier Universitaire de Québec Research Center, Université Laval, Québec, Quebec, Canada
| | - Nellie Dumont
- Medical Affairs and Innovation, Héma-Qubec, Québec, Quebec, Canada
| | - Yannick Doyon
- Centre Hospitalier Universitaire de Québec Research Center, Université Laval, Québec, Quebec, Canada
| | - Josée Laganière
- Medical Affairs and Innovation, Héma-Qubec, Québec, Quebec, Canada.
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10
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Jeon SB, Koh H, Han AR, Kim J, Lee S, Lee JH, Im SS, Yoon YS, Lee JH, Lee JY. Ferric citrate and apo-transferrin enable erythroblast maturation with β-globin from hemogenic endothelium. NPJ Regen Med 2023; 8:46. [PMID: 37626061 PMCID: PMC10457393 DOI: 10.1038/s41536-023-00320-4] [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] [Received: 11/22/2022] [Accepted: 08/08/2023] [Indexed: 08/27/2023] Open
Abstract
Red blood cell (RBC) generation from human pluripotent stem cells (PSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with β-globin expression in culture systems. During induction of β-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we describe a simple and reproducible culture system that can be used to generate erythroblasts with β-globin expression. We prepared a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium (HE). Floating erythroblasts derived from HE cells were primarily CD45+CD71+CD235a+ cells, and their number increased remarkably upon Fe treatment. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of β-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. The rapid maturation of these erythroblasts into RBCs was observed when injected in vivo, suggesting the development of RBCs that were ready to grow. Hence, induction of β-globin expression may be explained by the effects of ferric citrate that promote cell maturation by binding with soluble transferrin and entering the cells.Taken together, upon treatment with Fe, erythroblasts showed advanced maturity with a high transcription of β-globin. These findings can help devise a stable protocol for the generation of clinically applicable RBCs.
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Affiliation(s)
- Soo-Been Jeon
- CHA Advanced Research Institute, Bundang CHA Medical Center, CHA University, Seongnam, Kyunggi-do, 13488, South Korea
| | - Hyebin Koh
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Republic of Korea
| | - A-Reum Han
- CHA Advanced Research Institute, Bundang CHA Medical Center, CHA University, Seongnam, Kyunggi-do, 13488, South Korea
| | - Jieun Kim
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea
| | - Sunghun Lee
- CHA Advanced Research Institute, Bundang CHA Medical Center, CHA University, Seongnam, Kyunggi-do, 13488, South Korea
| | - Jae-Ho Lee
- Department of Physiology, Keimyung University School of Medicine, Daegu, 42601, Korea
| | - Seung-Soon Im
- Department of Physiology, Keimyung University School of Medicine, Daegu, 42601, Korea
| | - Young-Sup Yoon
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea
- Department of Medicine, Emory University, Atlanta, USA
| | - Jong-Hee Lee
- Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Republic of Korea.
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea.
| | - Ji Yoon Lee
- CHA Advanced Research Institute, Bundang CHA Medical Center, CHA University, Seongnam, Kyunggi-do, 13488, South Korea.
- Department of Biomedical Science, CHA University, Seongnam, Kyunggi-do, 13488, South Korea.
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.
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11
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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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12
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In Vitro Human Haematopoietic Stem Cell Expansion and Differentiation. Cells 2023; 12:cells12060896. [PMID: 36980237 PMCID: PMC10046976 DOI: 10.3390/cells12060896] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2023] [Revised: 03/08/2023] [Accepted: 03/09/2023] [Indexed: 03/17/2023] Open
Abstract
The haematopoietic system plays an essential role in our health and survival. It is comprised of a range of mature blood and immune cell types, including oxygen-carrying erythrocytes, platelet-producing megakaryocytes and infection-fighting myeloid and lymphoid cells. Self-renewing multipotent haematopoietic stem cells (HSCs) and a range of intermediate haematopoietic progenitor cell types differentiate into these mature cell types to continuously support haematopoietic system homeostasis throughout life. This process of haematopoiesis is tightly regulated in vivo and primarily takes place in the bone marrow. Over the years, a range of in vitro culture systems have been developed, either to expand haematopoietic stem and progenitor cells or to differentiate them into the various haematopoietic lineages, based on the use of recombinant cytokines, co-culture systems and/or small molecules. These approaches provide important tractable models to study human haematopoiesis in vitro. Additionally, haematopoietic cell culture systems are being developed and clinical tested as a source of cell products for transplantation and transfusion medicine. This review discusses the in vitro culture protocols for human HSC expansion and differentiation, and summarises the key factors involved in these biological processes.
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13
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Boonpeng K, Ketprasit N, Palasuwan A, Kulkeaw K, Palasuwan D. Glucose-6-phosphate dehydrogenase is dispensable for human erythroid cell differentiation in vitro. Exp Hematol 2023; 121:18-29.e2. [PMID: 36801436 DOI: 10.1016/j.exphem.2023.02.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 02/07/2023] [Accepted: 02/08/2023] [Indexed: 02/20/2023]
Abstract
Glucose-6-phosphate dehydrogenase (G6PD) deficiency impairs cellular processes under oxidative stress. Individuals with severe G6PD deficiency still produce sufficient numbers of erythrocytes. Nevertheless, the G6PD independence of erythropoiesis remains questionable. This study elucidates the effects of G6PD deficiency on the generation of human erythrocytes. Peripheral blood-derived CD34-positive hematopoietic stem and progenitor cells (HSPCs) of human subjects with normal, moderate, and severe G6PD activities were cultured in two distinct phases: erythroid commitment and terminal differentiation. Regardless of G6PD deficiency, HSPCs were able to proliferate and differentiate into mature erythrocytes. There was no impairment in erythroid enucleation among the subjects with G6PD deficiency. To our knowledge, this study is the first report of effective erythropoiesis independent of G6PD deficiency. The evidence firmly indicates that the population with the G6PD variant could produce erythrocytes to an extent similar to that in healthy individuals.
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Affiliation(s)
- Kanyarat Boonpeng
- Programme in Clinical Hematology Sciences, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Nutpakal Ketprasit
- Oxidation in Red Cell Disorders Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Attakorn Palasuwan
- Oxidation in Red Cell Disorders Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Kasem Kulkeaw
- Siriraj Integrative Center for Neglected Parasitic Diseases, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
| | - Duangdao Palasuwan
- Oxidation in Red Cell Disorders Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
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14
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Lu Z, Xu G, Li Y, Lu C, Shen Y, Zhao B. Discovery of N-arylcinnamamides as novel erythroblast enucleation inducers. Bioorg Chem 2022; 128:106105. [PMID: 36031698 DOI: 10.1016/j.bioorg.2022.106105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 08/12/2022] [Accepted: 08/17/2022] [Indexed: 01/28/2023]
Abstract
Derivation of mature red blood cells (RBCs) from stem cells in vitro is a promising solution to the current shortage of blood supply, in which terminal enucleation is the rate-limiting step. Here we discovered two cinnamamides B8 and B16 showed potential activities of enhancing the enucleation of erythroblasts through the screening of "in-house" compound library. Subsequently, twenty-four N-arylcinnamamides were rationally designed and synthesized on the basis of the structure of B8 and B16, in which N-(9H-carbazol-2-yl)cinnamamide (KS-2) significantly elevated the percentage of reticulocytes in the cultured mouse fetal liver cells in vitro (relative enucleation = 2.43). The underlying mechanism of KS-2 in promoting mouse erythroid enucleation is accelerating the process of cell cycle exit via p53 activation in late stage erythrocytes. These results strongly suggest that compound KS-2 is worthy of further study as a potential erythrocyte enucleation inducer.
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Affiliation(s)
- Zhiyuan Lu
- Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, China
| | - Guangsen Xu
- Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China
| | - Yanxia Li
- Department of Pharmacology, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China
| | - Chunhua Lu
- Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; NMPA Key Laboratory for Technology Research and Evaluation of Drug Products, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China
| | - Yuemao Shen
- Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; NMPA Key Laboratory for Technology Research and Evaluation of Drug Products, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China
| | - Baobing Zhao
- Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; Department of Pharmacology, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; NMPA Key Laboratory for Technology Research and Evaluation of Drug Products, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China.
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15
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Soboleva S, Miharada K. Induction of enucleation in primary and immortalized erythroid cells. Int J Hematol 2022; 116:192-198. [PMID: 35610497 DOI: 10.1007/s12185-022-03386-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 05/02/2022] [Accepted: 05/06/2022] [Indexed: 10/18/2022]
Abstract
Enucleation is a crucial event during the erythropoiesis, implicating drastic morphologic and transcriptomic/proteomic changes. While many genes deletion lead to failed or impaired enucleation have been identified, directly triggering the erythroid maturation, particularly enucleation, is still challenging. Inducing enucleation at the desired timing is necessary to develop efficient methods to generate mature, fully functional red blood cells in vitro for future transfusion therapies. However, there are considerable differences between primary erythroid cells and cultured cell sources, particularly pluripotent stem cell-derived erythroid cells and immortalized erythroid cell lines. For instance, the difference in the proliferative status between those cell types could be a critical factor, as cell cycle exit is closely connected to the terminal maturation of primary. In this review, we will discuss previous findings on the enucleation machinery and current challengings to trigger the enucleation of infinite erythroid cell sources.
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Affiliation(s)
- Svetlana Soboleva
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Kenichi Miharada
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden. .,International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.
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16
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Soboleva S, Åkerstrand H, Miharada K. Transcriptomic analysis of functional diversity of human umbilical cord blood hematopoietic stem/progenitor cells in erythroid differentiation. Int J Hematol 2022; 115:481-488. [PMID: 35088351 DOI: 10.1007/s12185-022-03292-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 01/18/2022] [Accepted: 01/19/2022] [Indexed: 11/26/2022]
Abstract
Hematopoietic stem cells (HSC) give rise to all types of blood lineages, including red blood cells (RBC). Hematopoietic stem/progenitor cells (HSPC) are known to be functionally diverse in terms of their self-renewal potential and lineage output. Consequently, investigation of molecular heterogeneity in the differentiation potential of HSPC is vital to identify novel regulators that affect generation of specific cell types, especially RBC. Here, we compared the erythroid potential of CD34+ hematopoietic stem and progenitor cells from 50 different umbilical cord blood (UCB) donors and discovered that those donors gave rise to diverse frequencies of Glycophorin-A+ erythroid cells after in vitro differentiation, despite having similar frequencies of phenotypic HSC initially. RNA sequencing revealed that genes involved in G protein-coupled receptor (GPCR) signaling were significantly up-regulated in the high-erythroid output donors. When we chemically modified two main signaling elements in this pathway, adenylyl cyclase (AC) and phosphodiesterase (PDE), we observed that inhibition of PDE led to 10 times higher yield of Glycophorin-A+ cells than activation of AC. Our findings suggest that GPCR signaling, and particularly the cAMP-related pathway, contributes to the diversity of erythroid potential among UCB donors.
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Affiliation(s)
- Svetlana Soboleva
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Hugo Åkerstrand
- Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Kenichi Miharada
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.
- International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.
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17
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Soboleva S, Kurita R, Kajitani N, Åkerstrand H, Miharada K. Establishment of an immortalized human erythroid cell line sustaining differentiation potential without inducible gene expression system. Hum Cell 2021; 35:408-417. [PMID: 34817797 DOI: 10.1007/s13577-021-00652-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 11/17/2021] [Indexed: 11/30/2022]
Abstract
Ex vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential. Here, we report that erythroid cell lines established from human bone marrow using simple expression of HPV-E6/E7 are capable of normal erythroid differentiation, without turning gene expression off. Through simply changing cell culture conditions, a newly established cell line, Erythroid Line from Lund University (ELLU), is able to differentiate toward mature cells, including enucleated reticulocytes. ELLU is heterogeneous and, unexpectedly, clones expressing adult hemoglobin rapidly differentiate and produce fragile cells. Upon differentiation, other ELLU clones shift from fetal to adult hemoglobin expression, giving rise to more mature cells. Our findings propose that it is not necessary to employ gene induction systems to establish immortalized erythroid cell lines sustaining differentiation potential and describe novel cellular characteristics for desired functionally competent clones.
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Affiliation(s)
- Svetlana Soboleva
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Ryo Kurita
- Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan
| | - Naoko Kajitani
- Division of Medical Microbiology, Lund University, Lund, Sweden
| | - Hugo Åkerstrand
- Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Kenichi Miharada
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden. .,International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.
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18
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Wan G, Medina S, Zhang H, Pan R, Zhou X, Bolt AM, Luo L, Burchiel SW, Liu KJ. Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34 + cells and causes decreased levels of hemoglobin. Sci Rep 2021; 11:22121. [PMID: 34764389 PMCID: PMC8586241 DOI: 10.1038/s41598-021-01643-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Accepted: 11/01/2021] [Indexed: 02/02/2023] Open
Abstract
Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.
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Affiliation(s)
- Guanghua Wan
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Sebastian Medina
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
- Department of Biology, New Mexico Highlands University, Las Vegas, NM, 87701, USA
| | - Haikun Zhang
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Rong Pan
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Xixi Zhou
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Alicia M Bolt
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Li Luo
- Division of Epidemiology, Biostatistics and Preventive Medicine at the University of New Mexico, Albuquerque, NM, 87131, USA
| | - Scott W Burchiel
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA
| | - Ke Jian Liu
- Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM, 87131, USA.
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19
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Xie X, Yao H, Han X, Yue W, Pei X. Therapeutic use of red blood cells and platelets derived from human cord blood stem cells. Stem Cells Transl Med 2021; 10 Suppl 2:S48-S53. [PMID: 34724719 PMCID: PMC8560193 DOI: 10.1002/sctm.20-0517] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2020] [Revised: 05/15/2021] [Accepted: 06/06/2021] [Indexed: 12/19/2022] Open
Abstract
Red blood cells (RBCs) and platelets derived from stem cells are possible solutions to the increasing demand for blood transfusion. Based on the availability of stem cells, their relatively defined differentiation mechanisms, and the massive exploration of induction systems, the generation of RBCs or platelets in vitro from cord blood hematopoietic stem/progenitor cells (CB-HSPCs) has potential for clinical applications. However, information on the clinical translation of stem cell-derived RBCs and platelets in the literature and at the ClinicalTrials.gov website is very limited. The only clinical trial on cultured RBCs, which aimed to assess the lifespan of RBCs cultured in vivo, was reported by Luc Douay and colleagues. Of note, the cultured RBCs they used were derived from autologous peripheral blood HSPCs, and no cultured platelets have been applied clinically to date. However, CB-HSPC-derived megakaryocytes, platelet precursors, have been used in the treatment of thrombocytopenia. A successful phase I trial was reported, followed by phase II and III clinical trials conducted in China. In this review, the gap between the many basic studies and limited clinical trials on stem cell-derived RBCs and platelets is summarized. The possible reasons and solutions for this gap are discussed. Further technological improvements for blood cell expansion and maturation ex vivo and the establishment of biological standards for stem cell derivatives might help to facilitate the therapeutic applications of cultured RBCs and platelets derived from CB-HSPCs in the near future.
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Affiliation(s)
- Xiaoyan Xie
- Stem Cells and Regenerative Medicine LabInstitute of Health Service and Transfusion MedicineBeijingPeople's Republic of China
- South China Research Center for Stem Cell & Regenerative MedicineGuangzhouPeople's Republic of China
| | - Hailei Yao
- Stem Cells and Regenerative Medicine LabInstitute of Health Service and Transfusion MedicineBeijingPeople's Republic of China
- South China Research Center for Stem Cell & Regenerative MedicineGuangzhouPeople's Republic of China
| | - Xiaoyan Han
- National Institutes for Food and Drug ControlBeijingPeople's Republic of China
| | - Wen Yue
- Stem Cells and Regenerative Medicine LabInstitute of Health Service and Transfusion MedicineBeijingPeople's Republic of China
- South China Research Center for Stem Cell & Regenerative MedicineGuangzhouPeople's Republic of China
| | - Xuetao Pei
- Stem Cells and Regenerative Medicine LabInstitute of Health Service and Transfusion MedicineBeijingPeople's Republic of China
- South China Research Center for Stem Cell & Regenerative MedicineGuangzhouPeople's Republic of China
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20
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Cannon M, Phillips H, Smith S, Mitchell S, Landes K, Desai P, Byrd J, Lapalombella R. Red blood cells differentiated in vitro using sequential liquid and semi-solid culture as a pre-clinical model. Exp Hematol Oncol 2021; 10:50. [PMID: 34715921 PMCID: PMC8555309 DOI: 10.1186/s40164-021-00244-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Accepted: 10/21/2021] [Indexed: 11/10/2022] Open
Abstract
The in vitro erythrocyte differentiation model remains a strong, clinically relevant tool to model erythroid development in normal and disease related hematopoiesis. This model also has application to developing therapeutics for diseases related to red blood cells such as sickle cell anemia where targeting increased expression of fetal hemoglobin has been a major emphasis. Since the original protocol's publication in 2002, many groups have published modified methodologies to address issues in efficiency of maturation and terminal enucleation, as well as in scalability. While all reports have merit and show efficient enucleation, the methodologies used vary widely in technique and cytokine content. Additionally, despite the strengths in these methods, reproducibility of efficient differentiation to the point of differentiation is difficult. To address these limitations, we developed a streamlined process where total PBMCs are first primed using the original liquid culture expansion phase (published in 2002) before being differentiated with minimal input via standardized, commercially purchased semi-solid medium culture pre-supplemented with erythropoietin. Our data show this methodology to produce similar levels of CD235/CD71 positivity as previous methods but with enhanced CD235 solo positivity and evidence of enucleated cells in comparison with other widely used methods. Given the difficulty and wide variation in in vitro differentiation techniques, we present this methodology as a streamlined methodology for production of mature erythroid cells with minimal input using easily purchased reagents.
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Affiliation(s)
- Matthew Cannon
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - Hannah Phillips
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - Sidney Smith
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - Shaneice Mitchell
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - Kristina Landes
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - Payal Desai
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA
| | - John Byrd
- grid.261331.40000 0001 2285 7943Division of Hematology, The Ohio State University, Columbus, OH USA ,grid.261331.40000 0001 2285 7943College of Veterinary Medicine, The Ohio State University, Columbus, OH USA ,grid.261331.40000 0001 2285 7943Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH USA
| | - Rosa Lapalombella
- Division of Hematology, The Ohio State University, Columbus, OH, USA. .,The Ohio State University, Room 455C, OSUCCC Building, 410 West 12th Avenue, Columbus, OH, 43210, USA.
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21
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Industrially Compatible Transfusable iPSC-Derived RBCs: Progress, Challenges and Prospective Solutions. Int J Mol Sci 2021; 22:ijms22189808. [PMID: 34575977 PMCID: PMC8472628 DOI: 10.3390/ijms22189808] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2021] [Revised: 09/06/2021] [Accepted: 09/07/2021] [Indexed: 02/06/2023] Open
Abstract
Amidst the global shortfalls in blood supply, storage limitations of donor blood and the availability of potential blood substitutes for transfusion applications, society has pivoted towards in vitro generation of red blood cells (RBCs) as a means to solve these issues. Many conventional research studies over the past few decades have found success in differentiating hematopoietic stem and progenitor cells (HSPCs) from cord blood, adult bone marrow and peripheral blood sources. More recently, techniques that involve immortalization of erythroblast sources have also gained traction in tackling this problem. However, the RBCs generated from human induced pluripotent stem cells (hiPSCs) still remain as the most favorable solution due to many of its added advantages. In this review, we focus on the breakthroughs for high-density cultures of hiPSC-derived RBCs, and highlight the major challenges and prospective solutions throughout the whole process of erythropoiesis for hiPSC-derived RBCs. Furthermore, we elaborate on the recent advances and techniques used to achieve cost-effective, high-density cultures of GMP-compliant RBCs, and on their relevant novel applications after downstream processing and purification.
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22
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Kronstein-Wiedemann R, Thiel J, Tonn T. Blood Pharming – eine realistische Option? TRANSFUSIONSMEDIZIN 2021. [DOI: 10.1055/a-1342-0820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
ZusammenfassungDie Bluttransfusion ist ein wesentlicher und unersetzlicher Teil der modernen Medizin. Jedoch stellt vor allem bei Patienten mit sehr seltenen Blutgruppenkonstellationen der Mangel an Blutprodukten auch heute noch ein wichtiges Gesundheitsproblem weltweit dar. Um diesem Problem entgegenzutreten, versucht man seit einiger Zeit künstlich rote Blutzellen zu generieren. Diese haben potenzielle Vorteile gegenüber Spenderblut, wie z. B. ein verringertes Risiko für die Übertragung von Infektionskrankheiten. Diese Übersicht fasst die aktuellen Entwicklungen über den Prozess der Erythropoese, die Expansionsstrategien der erythrozytären Zellen, der verschiedenen Quellen für ex vivo expandierte Erythrozyten, die Hürden für die klinische Anwendung und die zukünftigen Möglichkeiten der Anwendung zusammen.
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Affiliation(s)
- Romy Kronstein-Wiedemann
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
| | - Jessica Thiel
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
| | - Torsten Tonn
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
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23
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Soboleva S, Kurita R, Ek F, Åkerstrand H, Silvério-Alves R, Olsson R, Nakamura Y, Miharada K. Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines. Commun Biol 2021; 4:677. [PMID: 34083702 PMCID: PMC8175573 DOI: 10.1038/s42003-021-02202-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Accepted: 05/11/2021] [Indexed: 11/09/2022] Open
Abstract
Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies. In an imaging-based screen of >3,300 compounds compounds, Soboleva et al identify HDAC inhibitors as mediators of erythroid cell enucleation. They further show that the erythroid-specific cell membrane protein, SPTA1, is downregulated in HDAC inhibited cells and that restoration of SPTA1 expression using CRISPR-activation partially rescues the fragility of cells, improving enucleation efficiency.
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Affiliation(s)
- Svetlana Soboleva
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Ryo Kurita
- Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan
| | - Fredrik Ek
- Chemical Biology and Therapeutics, Department of Experimental Medical Science, Lund University, Lund, Sweden
| | - Hugo Åkerstrand
- Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Rita Silvério-Alves
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.,Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden
| | - Roger Olsson
- Chemical Biology and Therapeutics, Department of Experimental Medical Science, Lund University, Lund, Sweden
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan
| | - Kenichi Miharada
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden. .,International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.
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24
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Bagchi A, Nath A, Thamodaran V, Ijee S, Palani D, Rajendiran V, Venkatesan V, Datari P, Pai AA, Janet NB, Balasubramanian P, Nakamura Y, Srivastava A, Mohankumar KM, Thangavel S, Velayudhan SR. Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells. Cells 2021; 10:523. [PMID: 33804564 PMCID: PMC7999632 DOI: 10.3390/cells10030523] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 02/08/2021] [Accepted: 02/19/2021] [Indexed: 02/04/2023] Open
Abstract
Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.
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Affiliation(s)
- Abhirup Bagchi
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Aneesha Nath
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Vasanth Thamodaran
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Smitha Ijee
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Dhavapriya Palani
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Vignesh Rajendiran
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Vigneshwaran Venkatesan
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
- Manipal Academy of Higher Education, Manipal 576104, Karnataka, India
| | - Phaneendra Datari
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
| | - Aswin Anand Pai
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
| | - Nancy Beryl Janet
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
| | - Poonkuzhali Balasubramanian
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research Center, Ibaraki 3050074, Japan;
| | - Alok Srivastava
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
| | - Kumarasamypet Murugesan Mohankumar
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Saravanabhavan Thangavel
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
| | - Shaji R. Velayudhan
- Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India; (A.B.); (A.N.); (V.T.); (S.I.); (D.P.); (V.R.); (V.V.); (A.S.); (K.M.M.); (S.T.)
- Department of Hematology, Christian Medical College, Vellore 632002, Tamil Nadu, India; (P.D.); (A.A.P.); (N.B.J.); (P.B.)
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25
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Liu S, Wu M, Lancelot M, Deng J, Gao Y, Roback JD, Chen T, Cheng L. BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells. Mol Ther 2021; 29:1918-1932. [PMID: 33484967 PMCID: PMC8116606 DOI: 10.1016/j.ymthe.2021.01.022] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Revised: 11/26/2020] [Accepted: 01/12/2021] [Indexed: 01/06/2023] Open
Abstract
Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.
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Affiliation(s)
- Senquan Liu
- Blood and Cell Therapy Institute, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Mengyao Wu
- Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Division of Hematology, Huashan Hospital of Fudan University, Shanghai 200040, China
| | - Moira Lancelot
- Center for Transfusion and Cellular Therapies, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Jiusheng Deng
- Center for Transfusion and Cellular Therapies, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Yongxing Gao
- Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - John D Roback
- Center for Transfusion and Cellular Therapies, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
| | - Tong Chen
- Division of Hematology, Huashan Hospital of Fudan University, Shanghai 200040, China.
| | - Linzhao Cheng
- Blood and Cell Therapy Institute, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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26
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Liu Q, Luo L, Ren C, Zou M, Yang S, Cai B, Wu L, Wang Y, Fu S, Hua X, Tang N, Huang S, Huang X, Xin W, Chen F, Zhang X. The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34 + cell erythropoiesis. Stem Cells 2020; 38:1492-1505. [PMID: 32871057 PMCID: PMC7693065 DOI: 10.1002/stem.3268] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2020] [Revised: 06/25/2020] [Accepted: 07/10/2020] [Indexed: 02/05/2023]
Abstract
As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood-borne infections since donated blood is the only source of RBCs. Although large-scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild-type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of β-thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood-derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell-cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.
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Affiliation(s)
- Qian Liu
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
- Shantou University Medical CollegeShantouPeople's Republic of China
| | - Linhong Luo
- Department of Obstetrics and GynecologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Chunhong Ren
- Department of International Medical ServiceThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Muping Zou
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Siqin Yang
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Bozhi Cai
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Libiao Wu
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Yunsheng Wang
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Shan Fu
- Shantou University Medical CollegeShantouPeople's Republic of China
| | - Xu Hua
- Shantou University Medical CollegeShantouPeople's Republic of China
| | - Nianping Tang
- Shantou University Medical CollegeShantouPeople's Republic of China
| | - Shiping Huang
- Shantou University Medical CollegeShantouPeople's Republic of China
| | - Xianxi Huang
- Intensive Care Unit, The First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Wen Xin
- Beijing TransGen Biotech Co., Ltd.BeijingPeople's Republic of China
| | - Feiheng Chen
- Department of HematologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
| | - Xin Zhang
- Laboratory of Molecular CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
- Shantou University Medical CollegeShantouPeople's Republic of China
- Department of CardiologyThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
- Laboratory of Medical Molecular ImagingThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople's Republic of China
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27
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Zeming KK, Sato Y, Yin L, Huang NJ, Wong LH, Loo HL, Lim YB, Lim CT, Chen J, Preiser PR, Han J. Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture. LAB ON A CHIP 2020; 20:3445-3460. [PMID: 32793940 DOI: 10.1039/c9lc01128e] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost. DFF sorting of reticulocytes from whole erythroid culture showed a 2.4-fold increase in cell recovery compared to FACS albeit with a lower purity; DLD sorting showed comparable cell recovery and purity with FACS using an inverse-L pillar structure to emphasize size and deformability sorting of reticulocytes. The viability and functional assurance of purified reticulocytes showed conserved cell deformability and supported the propagation of malaria parasites. Collectively, our study on label-free RBCs isolation represents a significant technical advancement towards developing in vitro generated viable human RBCs, opening opportunities for close-loop cell manufacturing, downstream therapeutic and research purposes.
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Affiliation(s)
- Kerwin Kwek Zeming
- Critical Analytics for Manufacturing of Personalized Medicine, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology, 138602, Singapore
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28
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Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells. Blood Adv 2020; 3:3337-3350. [PMID: 31698463 DOI: 10.1182/bloodadvances.2019000689] [Citation(s) in RCA: 57] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Accepted: 09/09/2019] [Indexed: 12/11/2022] Open
Abstract
Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+ isolation, and a 3 × 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117-DRAQ5- RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.
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29
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Reddy OL, Savani BN, Stroncek DF, Panch SR. Advances in gene therapy for hematologic disease and considerations for transfusion medicine. Semin Hematol 2020; 57:83-91. [PMID: 32892847 DOI: 10.1053/j.seminhematol.2020.07.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Indexed: 12/26/2022]
Abstract
As the list of regulatory agency-approved gene therapies grows, these products are now in the therapeutic spotlight with the potential to cure or dramatically alleviate several benign and malignant hematologic diseases. The mechanisms for gene manipulation are diverse, and include the use of a variety of cell sources and both viral vector- and nuclease-based targeted approaches. Gene editing has also reached the realm of blood component therapy and testing, where cultured products are being developed to improve transfusion support for individuals with rare blood types. In this review, we summarize the milestones in the development of gene therapies for hematologic diseases, mechanisms for gene manipulation, and implications for transfusion medicine and blood centers as these therapies continue to advance and grow.
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Affiliation(s)
- Opal L Reddy
- Center for Cellular Engineering, National institutes of Health, Clinical Center, Bethesda, Maryland
| | - Bipin N Savani
- Vanderbilt University Medical Center, Nashville, Tennessee
| | - David F Stroncek
- Center for Cellular Engineering, National institutes of Health, Clinical Center, Bethesda, Maryland
| | - Sandhya R Panch
- Center for Cellular Engineering, National institutes of Health, Clinical Center, Bethesda, Maryland.
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30
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Wu K, Tao J, Liao Q, Chen S, Wan W. Intracellular microtubules as nano-scaffolding template self-assembles with conductive carbon nanotubes for biomedical device. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 113:110971. [PMID: 32487389 DOI: 10.1016/j.msec.2020.110971] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 02/12/2020] [Accepted: 04/14/2020] [Indexed: 11/17/2022]
Abstract
Cellular bilayer and its membrane have been mimicked and for decades, e.g., to synthesize amphiphilic carriers for controlled release. Here we report using nanosized cellular microtubules (MT) as scaffolding template and amphiphilic cytomembrane fragment to self-assemble with hydrophobic carbon nanotubes (MWNT). The hybrid was then cross-linked to form a conductive scaffold. Polyaniline (PANI) was finally added to the nanocomposite to enhance conductivity. Being an electrode, the obtained cell-based conductive gel raise interfacial surface area, increase the conductivity of the material, and enhance the energy density and power density of the material with a relatively low MWNTs concentration (less than 4.8 wt%). The cell-based supercapacitor reaches a specific capacitance of 209.2 F/g and thus the fabricated cell-based electrode achieves a conductivity of 38Scm-1. The cellular electric device exhibits great potential for future implantable bio-device and bio-electronic interface applications.
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Affiliation(s)
- Kai Wu
- Department of Orthopaedic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Jun Tao
- Department of Orthopaedic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Qi Liao
- Department of Orthopaedic Surgery, The Third Affiliated Hospital of Nanchang University, Nanchang 330008, China
| | - Shixuan Chen
- Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, NE 68198, USA..
| | - Wenbing Wan
- Department of Orthopaedic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China.
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31
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Zamani M, Yaghoubi Y, Naimi A, Hassanzadeh A, Pourakbari R, Aghebati-Maleki L, Motavalli R, Aghlmandi A, Mehdizadeh A, Nazari M, Yousefi M, Movassaghpour AA. Humanized Culture Medium for Clinical-Grade Generation of Erythroid Cells from Umbilical Cord Blood CD34 + Cells. Adv Pharm Bull 2020; 11:335-342. [PMID: 33880356 PMCID: PMC8046389 DOI: 10.34172/apb.2021.031] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2020] [Revised: 02/03/2020] [Accepted: 02/29/2020] [Indexed: 12/30/2022] Open
Abstract
Purpose: Transfusion of red blood cells (RBCs) is a supportive and common treatment in surgical care, trauma, and anemia. However, in vivo production of RBC seems to be a suitable alternative for blood transfusions due to the limitation of blood resources, the possibility of disease transmission, immune reactions, and the presence of rare blood groups. Cell cultures require serum-free or culture media supplemented with highly expensive animal serum, which can transmit xenoviruses. Platelet lysate (PL) can be considered as a suitable alternative containing a high level of growth factors and a low production cost. Methods: Three-step culture media supplemented with PL or fetal bovine serum (FBS) were used for proliferation and differentiation of CD34+ umbilical cord blood stem cells to erythrocytes in co-culture with bone marrow mesenchymal stem cells (BM-MSCs). The cells were cultivated for 15 days and cell proliferation and expansion were assessed using cell counts at different days. Erythroid differentiation genes, CD71 and glycophorin A expression levels were evaluated. Results: Maximum hematopoietic stem cells (HSCs) proliferation was observed on day 15 in PL-containing medium (99±17×103-fold). Gene expression and surface markers showed higher differentiation of cells in PL-containing medium. Conclusion: The results of this study indicate that PL can enhance erythroid proliferation and differentiation of CD34+ HSCs. PL can also be used as a proper alternative for FBS in the culture medium and HSCs differentiation.
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Affiliation(s)
- Majid Zamani
- Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Yoda Yaghoubi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Adel Naimi
- Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran
| | - Ali Hassanzadeh
- Department of Tissue Engineering and Applied Cell Sciences, Tehran University of Medical Sciences, Tehran, Iran
| | - Ramin Pourakbari
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Roza Motavalli
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Afsoon Aghlmandi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Amir Mehdizadeh
- Endocrine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mehdi Nazari
- Department of Anesthesiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mehdi Yousefi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ali Akbar Movassaghpour
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
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32
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Guzniczak E, Otto O, Whyte G, Chandra T, Robertson NA, Willoughby N, Jimenez M, Bridle H. Purifying stem cell-derived red blood cells: a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration. Biotechnol Bioeng 2020; 117:2032-2045. [PMID: 32100873 PMCID: PMC7383897 DOI: 10.1002/bit.27319] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2019] [Revised: 02/17/2020] [Accepted: 02/24/2020] [Indexed: 02/06/2023]
Abstract
Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.
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Affiliation(s)
- Ewa Guzniczak
- Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science, Heriot-Watt University, Edinburgh, Scotland
| | - Oliver Otto
- Centre for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.,Deutsches Zentrum für Herz-Kreislaufforschung, Partner Site Greifswald, Greifswald, Germany
| | - Graeme Whyte
- Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science, Heriot-Watt University, Edinburgh, Scotland
| | - Tamir Chandra
- MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh, Western General Hospital, Edinburgh, Scotland
| | - Neil A Robertson
- MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh, Western General Hospital, Edinburgh, Scotland
| | - Nik Willoughby
- Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science, Heriot-Watt University, Edinburgh, Scotland
| | - Melanie Jimenez
- Biomedical Engineering Division, James Watt School of Engineering, University of Glasgow, Glasgow, Scotland
| | - Helen Bridle
- Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science, Heriot-Watt University, Edinburgh, Scotland
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33
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Kuhikar R, Khan N, Philip J, Melinkeri S, Kale V, Limaye L. Transforming growth factor β1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy. Stem Cell Res Ther 2020; 11:71. [PMID: 32075694 PMCID: PMC7029523 DOI: 10.1186/s13287-020-01603-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Revised: 01/31/2020] [Accepted: 02/12/2020] [Indexed: 12/14/2022] Open
Abstract
Background Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21 days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor β1 (TGF-β1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration it stimulates the HSCs growth. At high concentration, it also inhibits erythropoiesis but accelerates terminal erythroid differentiation of cell lines and erythroid progenitors. Here we examined whether the use of low concentration of TGF-β1 would be beneficial for increasing RBC production by stimulating HSC growth and also supporting erythroid differentiation. Such a strategy could make RBC production in vitro more efficient and cost-effective for clinical applications. Methods HSCs isolated from Apheresis samples were differentiated into mature RBCs by the sequential addition of specific combinations of growth factors for 21 days. In the control set, only EPO (3 IU/ml) was added whereas, in the test set, TGF-β1 at a concentration of 10 pg/ml was added along with EPO (3 IU/ml) from day 0. Results We found that a low concentration of TGF-β1 has no inhibitory effect on the proliferation of the early stages of erythropoiesis. Additionally, it significantly accelerates terminal stages of erythroid differentiation by promoting BNIP3L/NIX-mediated mitophagy. Conclusions Incorporation of TGF-β1 at 10 pg/ml concentration in the differentiation medium accelerates the in vitro erythropoiesis process by 3 days. This finding could have potential applications in transfusion medicine. Electronic supplementary material The online version of this article (10.1186/s13287-020-01603-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Rutuja Kuhikar
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India
| | - Nikhat Khan
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India
| | | | - Sameer Melinkeri
- Blood and Marrow Transplant Unit, Deenanath Mangeshkar Hospital, Erandawne, Pune, 411004, India
| | - Vaijayanti Kale
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India.,Symbiosis Centre for Stem Cell research, School of Biological Sciences, Symbiosis International (Deemed University), Lavale, Pune, 412115, India
| | - Lalita Limaye
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India.
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34
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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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35
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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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36
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Zingariello M, Bardelli C, Sancillo L, Ciaffoni F, Genova ML, Girelli G, Migliaccio AR. Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins. Front Physiol 2019; 10:281. [PMID: 31019464 PMCID: PMC6458278 DOI: 10.3389/fphys.2019.00281] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2019] [Accepted: 03/04/2019] [Indexed: 02/04/2023] Open
Abstract
Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo. The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2-3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs.
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Affiliation(s)
- Maria Zingariello
- Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, University Campus Bio-Medico, Rome, Italy
| | - Claudio Bardelli
- Department of Biomedical and NeuroMotor Sciences, Alma Mater Studiorum University, Bologna, Italy
| | - Laura Sancillo
- Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, University Campus Bio-Medico, Rome, Italy
| | | | - Maria Luisa Genova
- Department of Biomedical and NeuroMotor Sciences, Alma Mater Studiorum University, Bologna, Italy
| | | | - Anna Rita Migliaccio
- Department of Biomedical and NeuroMotor Sciences, Alma Mater Studiorum University, Bologna, Italy
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37
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Kim SH, Lee EM, Han SY, Choi HS, Ryu KY, Baek EJ. Improvement of Red Blood Cell MaturationIn Vitroby Serum-Free Medium Optimization. Tissue Eng Part C Methods 2019; 25:232-242. [DOI: 10.1089/ten.tec.2019.0023] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Affiliation(s)
- Seo Hui Kim
- Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
| | - Eun Mi Lee
- Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
| | - So Yeon Han
- Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
| | - Hye Sook Choi
- Department of Laboratory Medicine, College of Medicine, Hanyang University, Seoul, Republic of Korea
| | - Ki Young Ryu
- Departmemt of Obstetrics and Gynecology, College of Medicine, Hanyang University, Seoul, Republic of Korea
| | - Eun Jung Baek
- Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea
- Department of Laboratory Medicine, College of Medicine, Hanyang University, Seoul, Republic of Korea
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38
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Christaki EE, Politou M, Antonelou M, Athanasopoulos A, Simantirakis E, Seghatchian J, Vassilopoulos G. Ex vivo generation of transfusable red blood cells from various stem cell sources: A concise revisit of where we are now. Transfus Apher Sci 2019; 58:108-112. [DOI: 10.1016/j.transci.2018.12.015] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Poldee S, Metheetrairut C, Nugoolsuksiri S, Frayne J, Trakarnsanga K. Optimization of an erythroid culture system to reduce the cost of in vitro production of red blood cells. MethodsX 2018; 5:1626-1632. [PMID: 30560064 PMCID: PMC6290122 DOI: 10.1016/j.mex.2018.11.018] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Accepted: 11/28/2018] [Indexed: 11/30/2022] Open
Abstract
In vitro generation of red blood cells has become a goal for scientists globally. Directly, in vitro-generated red blood cells (RBCs) may close the gap between blood supply obtained through blood donation and high demand for therapeutic uses. In addition, the cells obtained can be used as a model for haematologic disorders to allow the study of their pathophysiology and novel treatment discovery. For those reasons, a number of RBC culture systems have been established and shown to be successful; however, the cost of each millilitre of packed RBC is still extremely high. In order to reduce the cost, we aim to see if we can reduce the number of factors used in the existing culture system. In this study, we examined how well haematopoietic stem cells proliferate and differentiate into mature red blood cells with modified culture system.
Absence of extra heparin or insulin or both from the erythroid differentiation media did not affect haematopoietic stem cell proliferation and differentiation. Therefore, we show that the cost and complexity of erythroid culture can be reduced, which may improve the feasibility of in vitro generation of red blood cells.
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Affiliation(s)
- Saiphon Poldee
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Chanatip Metheetrairut
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Sutthinee Nugoolsuksiri
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Jan Frayne
- School of Biochemistry, Faculty of Biomedical Sciences, University of Bristol, Bristol, BS8 1TD, UK
| | - Kongtana Trakarnsanga
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.,School of Biochemistry, Faculty of Biomedical Sciences, University of Bristol, Bristol, BS8 1TD, UK
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40
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Sun S, Peng Y, Liu J. Research advances in erythrocyte regeneration sources and methods in vitro. CELL REGENERATION 2018; 7:45-49. [PMID: 30671230 PMCID: PMC6326244 DOI: 10.1016/j.cr.2018.10.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 10/22/2018] [Accepted: 10/26/2018] [Indexed: 01/04/2023]
Abstract
Erythrocytes (red blood cells, RBCs) facilitate gas exchange in the lungs and transport oxygen to the tissues. The human body must maintain erythrocyte regeneration to support metabolically active cells and tissues. In many hematological diseases, erythrocyte regeneration is impaired. Researchers have studied erythrocyte regeneration for many years both in vivo and in vitro. In this review, we summarize the sources and main culture methods for generating mature and functional red blood cells in vitro. Hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are classic sources for erythrocyte regeneration. In addition, alternative sources such as immortalized adult human erythroid cell lines and transformed fibroblasts have also been generated and have produced functional red blood cells. The culture systems for erythrocytes differ among laboratories. Researchers hope that improvements in culture techniques may contribute to improved RBC outcomes for blood transfusions, drug delivery and the treatment of hematological diseases.
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Affiliation(s)
- Shuming Sun
- Molecular Biology Research Center & Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China.,Erythropoiesis Research Center, Central South University, Changsha 410078, China
| | - Yuanliang Peng
- Molecular Biology Research Center & Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China.,Erythropoiesis Research Center, Central South University, Changsha 410078, China
| | - Jing Liu
- Molecular Biology Research Center & Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China.,Erythropoiesis Research Center, Central South University, Changsha 410078, China
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41
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Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source. Exp Hematol 2018; 69:11-16. [PMID: 30326248 DOI: 10.1016/j.exphem.2018.10.005] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2018] [Revised: 10/09/2018] [Accepted: 10/10/2018] [Indexed: 12/20/2022]
Abstract
Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.
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42
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Lee E, Sivalingam J, Lim ZR, Chia G, Shi LG, Roberts M, Loh YH, Reuveny S, Oh SKW. Review: In vitro generation of red blood cells for transfusion medicine: Progress, prospects and challenges. Biotechnol Adv 2018; 36:2118-2128. [PMID: 30273713 DOI: 10.1016/j.biotechadv.2018.09.006] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 09/19/2018] [Accepted: 09/27/2018] [Indexed: 02/07/2023]
Abstract
In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.
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Affiliation(s)
- Esmond Lee
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Palo Alto, CA 94305, USA.
| | - Jaichandran Sivalingam
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore.
| | - Zhong Ri Lim
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore
| | - Gloryn Chia
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore
| | - Low Gin Shi
- Brilliant Research Pte. Ltd, Singapore 139955, Republic of Singapore
| | - Mackenna Roberts
- Oxford-University College London Centre for the Advancement of Sustainable Medical Innovation, University of Oxford, UK
| | - Yuin-Han Loh
- Institute of Molecular and Cellular Biology, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Republic of Singapore
| | - Shaul Reuveny
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore
| | - Steve Kah-Weng Oh
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Republic of Singapore
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43
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Alattar N, Daud H, Al-Majmaie R, Zeulla D, Al-Rubeai M, Rice JH. Surface-enhanced Raman scattering for rapid hematopoietic stem cell differentiation analysis. APPLIED OPTICS 2018; 57:E184-E189. [PMID: 30117870 DOI: 10.1364/ao.57.00e184] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Accepted: 06/29/2018] [Indexed: 05/27/2023]
Abstract
Raman-spectroscopy-based methods, such as surface-enhanced Raman spectroscopy, are a well-evolved method to molecular fingerprint cell types. Here we demonstrate that surface-enhanced Raman spectroscopy can enable us to distinguish cell development stages of bone marrow hematopoietic stem cells towards red blood cells through the identification of specific surface-enhanced Raman spectroscopy biomarkers. The approach taken here is to allow cells to take in gold nanoparticles as Raman enhancement platforms for kinetic structural observations presented here through the view of the multidimensional parameter contribution, thereby enabling profiling of bone marrow hematopoietic stem cells acquired from proliferation (stage one), differentiation (stage two), and mature red blood cells (stage three).
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44
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Ovchynnikova E, Aglialoro F, von Lindern M, van den Akker E. The Shape Shifting Story of Reticulocyte Maturation. Front Physiol 2018; 9:829. [PMID: 30050448 PMCID: PMC6050374 DOI: 10.3389/fphys.2018.00829] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Accepted: 06/12/2018] [Indexed: 12/11/2022] Open
Abstract
The final steps of erythropoiesis involve unique cellular processes including enucleation and reorganization of membrane proteins and the cytoskeleton to produce biconcave erythrocytes. Surprisingly this process is still poorly understood. In vitro erythropoiesis protocols currently produce reticulocytes rather than biconcave erythrocytes. In addition, immortalized lines and iPSC-derived erythroid cell suffer from low enucleation and suboptimal final maturation potential. In light of the increasing prospect to use in vitro produced erythrocytes as (personalized) transfusion products or as therapeutic delivery agents, the mechanisms driving this last step of erythropoiesis are in dire need of resolving. Here we review the elusive last steps of reticulocyte maturation with an emphasis on protein sorting during the defining steps of reticulocyte formation during enucleation and maturation.
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Affiliation(s)
- Elina Ovchynnikova
- Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands.,Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
| | - Francesca Aglialoro
- Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands.,Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
| | - Marieke von Lindern
- Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands.,Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
| | - Emile van den Akker
- Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands.,Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
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45
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Abstract
Blood transfusions are often essential for treatment of severe anaemia and pregnancy complications. The unavailability of blood is a medical concern, especially in developing countries. New sources of red blood cells (RBC) are under investigation. Several studies have attempted to produce functional RBC from CD34+ haematopoietic stem cells (HSC) isolated from peripheral blood and umbilical cord blood, from embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). A recent article published in Nature Communications describes a novel model for generating RBC from a stable erythroid cell line obtained from bone marrow CD34+ haematopoietic stem cells (HSC). The cells generated by this method are phenotypically and functionally adult RBC, that resemble very well the donor RBC. In vivo experiments confirmed no difference in the survival of these RBC and donor RBC. The study therefore highlights that this immortalized line is a promising new source of adult RBC.
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Affiliation(s)
- Maria Teresa Esposito
- Department of Life Sciences, University of Roehampton, Whiteland College, London, SW15 4JD UK
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46
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Minetti G, Achilli C, Perotti C, Ciana A. Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell. Front Physiol 2018; 9:286. [PMID: 29632498 PMCID: PMC5879444 DOI: 10.3389/fphys.2018.00286] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Accepted: 03/12/2018] [Indexed: 12/11/2022] Open
Abstract
Within the context of erythropoiesis and the possibility of producing artificial red blood cells (RBCs) in vitro, a most critical step is the final differentiation of enucleated erythroblasts, or reticulocytes, to a fully mature biconcave discocyte, the RBC. Reviewed here is the current knowledge about this fundamental maturational process. By combining literature data with our own experimental evidence we propose that the early phase in the maturation of reticulocytes to RBCs is driven by a membrane raft-based mechanism for the sorting of disposable membrane proteins, mostly the no longer needed transferrin receptor (TfR), to the multivesicular endosome (MVE) as cargo of intraluminal vesicles that are subsequently exocytosed as exosomes, consistently with the seminal and original observation of Johnstone and collaborators of more than 30 years ago (Pan BT, Johnstone RM. Cell. 1983;33:967-978). According to a strikingly selective sorting process, the TfR becomes cargo destined to exocytosis while other molecules, including the most abundant RBC transmembrane protein, band 3, are completely retained in the cell membrane. It is also proposed that while this process could be operating in the early maturational steps in the bone marrow, additional mechanism(s) must be at play for the final removal of the excess reticulocyte membrane that is observed to occur in the circulation. This processing will most likely require the intervention of the spleen, whose function is also necessary for the continuous remodeling of the RBC membrane all along this cell's circulatory life.
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Affiliation(s)
- Giampaolo Minetti
- Laboratori di Biochimica, Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia, Pavia, Italy
| | - Cesare Achilli
- Laboratori di Biochimica, Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia, Pavia, Italy
| | - Cesare Perotti
- Servizio Immunoematologia e Medicina Trasfusionale, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
| | - Annarita Ciana
- Laboratori di Biochimica, Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia, Pavia, Italy
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47
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Severn CE, Toye AM. The challenge of growing enough reticulocytes for transfusion. ACTA ACUST UNITED AC 2017. [DOI: 10.1111/voxs.12374] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Affiliation(s)
- C. E. Severn
- School of Biochemistry; Biomedical Sciences Building; University Walk; University of Bristol; Bristol UK
- National Institute for Health Research (NIHR) Blood and Transplant Research Unit; University of Bristol; Bristol UK
| | - A. M. Toye
- School of Biochemistry; Biomedical Sciences Building; University Walk; University of Bristol; Bristol UK
- National Institute for Health Research (NIHR) Blood and Transplant Research Unit; University of Bristol; Bristol UK
- Bristol Institute of Transfusion Sciences; NHSBT Filton; Bristol UK
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48
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Cappellino LA, Kratje RB, Etcheverrigaray M, Prieto CC. Strategy for erythroid differentiation in ex vivo cultures: Lentiviral genetic modification of human hematopoietic stem cells with erythropoietin gene. J Biosci Bioeng 2017; 124:591-598. [PMID: 28688754 DOI: 10.1016/j.jbiosc.2017.06.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2016] [Revised: 06/02/2017] [Accepted: 06/15/2017] [Indexed: 01/04/2023]
Abstract
If cultured in appropriate conditions, such as supplementing culture media with costly cytokines and growth factors, hematopoietic stem/progenitor cells (HSPCs) from different origins have shown to be an adequate source of erythroid cells. This requirement turns erythroid cells production into a complicated process to be scaled-up for future applications. The aim of our work was to genetically modify HSPCs with human erythropoietin (hEPO) sequence by lentiviral transgenesis in order for cells to secrete the hormone into the culture medium. Initially, we evaluated erythroid differentiation in colony forming units (CFU) assays and further analyzed cell expansion and erythroid differentiation throughout time in suspension cultures by flow cytometry and May-Grünwald-Giemsa staining. Additionally, we studied hEPO production and its isoforms profile. The different assessment approaches demonstrated erythroid differentiation, which was attributed to the hEPO secreted by the HSPCs. Our data demonstrate that it is possible to develop culture systems in which recombinant HSPCs are self-suppliers of hEPO. This feature makes our strategy attractive to be applied in biotechnological production processes of erythroid cells that are currently under development.
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Affiliation(s)
- Luisina A Cappellino
- UNL, CONICET, Cell Culture Laboratory, FBCB, Edificio FBCB-Ciudad Universitaria UNL, C.C. 242. (S3000ZAA), Santa Fe, Argentina
| | - Ricardo B Kratje
- UNL, CONICET, Cell Culture Laboratory, FBCB, Edificio FBCB-Ciudad Universitaria UNL, C.C. 242. (S3000ZAA), Santa Fe, Argentina
| | - Marina Etcheverrigaray
- UNL, CONICET, Cell Culture Laboratory, FBCB, Edificio FBCB-Ciudad Universitaria UNL, C.C. 242. (S3000ZAA), Santa Fe, Argentina
| | - Claudio C Prieto
- UNL, Cell Culture Laboratory, FBCB, Edificio FBCB-Ciudad Universitaria UNL, C.C. 242. (S3000ZAA), Santa Fe, Argentina.
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49
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Zhang Y, Wang C, Wang L, Shen B, Guan X, Tian J, Ren Z, Ding X, Ma Y, Dai W, Jiang Y. Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34 + Cells. Stem Cells Transl Med 2017; 6:1698-1709. [PMID: 28618139 PMCID: PMC5689780 DOI: 10.1002/sctm.17-0057] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2017] [Accepted: 05/08/2017] [Indexed: 12/17/2022] Open
Abstract
The ex vivo generation of human red blood cells on a large scale from hematopoietic stem and progenitor cells has been considered as a potential method to overcome blood supply shortages. Here, we report that functional human erythrocytes can be efficiently produced from cord blood (CB) CD34+ cells using a bottle turning device culture system. Safety and efficiency studies were performed in murine and nonhuman primate (NHP) models. With the selected optimized culture conditions, one human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1% ± 6.2% and 50% ± 5.7% (mean ± SD) for CD235a+ cells and enucleated cells, respectively. The yield of erythrocytes from one CB unit (5 million CD34+ cells) could be, in theory, equivalent to 500 blood transfusion units in clinical application. Moreover, induced human erythrocytes had normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model. In NHP model, xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia. These findings suggested that the ex vivo-generated erythrocytes could be an alternative blood source for traditional transfusion products in the clinic. Stem Cells Translational Medicine 2017;6:1698-1709.
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Affiliation(s)
- Yu Zhang
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
| | - Chen Wang
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,Biopharmagen Corp, Suzhou, China
| | - Lan Wang
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
| | - Bin Shen
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
| | - Xin Guan
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
| | - Jing Tian
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
| | - Zhihua Ren
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,Biopharmagen Corp, Suzhou, China
| | - Xinxin Ding
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,College of Nanoscale Science, SUNY Polytechnic Institute, Albany, New York, USA
| | - Yupo Ma
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,Department of Pathology, School of Medicine, The State University of New York at Stony Brook, Stony Brook, New York, USA
| | - Wei Dai
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,Department of Environment Medicine, New York University Langone Medical center, Tuxedo, New York, USA
| | - Yongping Jiang
- Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.,Biopharmagen Corp, Suzhou, China
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50
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Lau SX, Leong YY, Ng WH, Ng AWP, Ismail IS, Yusoff NM, Ramasamy R, Tan JJ. Human mesenchymal stem cells promote CD34 + hematopoietic stem cell proliferation with preserved red blood cell differentiation capacity. Cell Biol Int 2017; 41:697-704. [PMID: 28403524 DOI: 10.1002/cbin.10774] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 03/08/2017] [Indexed: 11/11/2022]
Abstract
Studies showed that co-transplantation of mesenchymal stem cells (MSCs) and cord blood-derived CD34+ hematopoietic stem cells (HSCs) offered greater therapeutic effects but little is known regarding the effects of human Wharton's jelly derived MSCs on HSC expansion and red blood cell (RBC) generation in vitro. This study aimed to investigate the effects of MSCs on HSC expansion and differentiation. HSCs were co-cultured with MSCs or with 10% MSCs-derived conditioned medium, with HSCs cultured under standard medium served as a control. Cell expansion rates, number of mononuclear cell post-expansion and number of enucleated cells post-differentiation were evaluated. HSCs showed superior proliferation in the presence of MSC with mean expansion rate of 3.5 × 108 ± 1.8 × 107 after day 7 compared to the conditioned medium and the control group (8.9 × 107 ± 1.1 × 108 and 7.0 × 107 ± 3.3 × 106 respectively, P < 0.001). Although no significant differences in RBC differentiation were observed between groups at passage IV, the number of enucleated cell was greater compared to earlier passages, indicating successful RBC differentiation. Cord blood-derived CD34+ HSCs can be greatly expanded by co-culturing with MSCs without affecting the RBC differentiation capability, suggesting the importance of direct MSC-HSCs contact in HSC expansion and RBC differentiation.
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Affiliation(s)
- Show Xuan Lau
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Yin Yee Leong
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Wai Hoe Ng
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Albert Wee Po Ng
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Ida Shazrina Ismail
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Narazah Mohd Yusoff
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
| | - Rajesh Ramasamy
- Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, 43400, Selangor Darul Ehsan, Malaysia
| | - Jun Jie Tan
- Advanced Medical and Dental Institute, UniversitiSains Malaysia, Bertam 13200 Kepala Batas, Penang, Malaysia
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