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Budagova T, Efremova A, Usman N, Mokrousova D, Goldshtein D. Differentiating Induced Pluripotent Stem Cells into Natural Killer Cells for Adoptive Cell Immunotherapies-Comparative Characterization of Current Protocols. Int J Mol Sci 2025; 26:1107. [PMID: 39940874 PMCID: PMC11816922 DOI: 10.3390/ijms26031107] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 01/23/2025] [Accepted: 01/23/2025] [Indexed: 02/16/2025] Open
Abstract
Cancers constitute a leading cause of mortality. Chimeric antigen receptor (CAR) cell therapies provide breakthrough solutions for various cancers while posing considerable risks of immunological side reactions. Of various cytotoxic lymphocyte subsets, natural killer (NK) cells are considered the least immunogenic. Obtaining viable NK cells with stable phenotypes in quantities sufficient for modification is technologically challenging. The candidate sources include primary mononuclear cell cultures and immortalized NK cell lines; alternatively, the clinical-grade NK cells can be differentiated from induced pluripotent stem cells (iPSCs) by a good manufacturing practice (GMP)-compatible xeno-free protocol. In this review, we analyze existing protocols for targeted differentiation of human iPSCs into NK cells with a focus on xeno-free requirements.
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Affiliation(s)
- Tatiana Budagova
- Research Centre for Medical Genetics, Moskvorechye Str. 1, Moscow 115522, Russia; (T.B.); (D.M.); (D.G.)
| | - Anna Efremova
- Research Centre for Medical Genetics, Moskvorechye Str. 1, Moscow 115522, Russia; (T.B.); (D.M.); (D.G.)
- Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia, Miklukho-Maklaya Str. 6, Moscow 117198, Russia
| | - Natalia Usman
- Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Samory Mashela Str. 1, Moscow 117198, Russia;
| | - Diana Mokrousova
- Research Centre for Medical Genetics, Moskvorechye Str. 1, Moscow 115522, Russia; (T.B.); (D.M.); (D.G.)
| | - Dmitry Goldshtein
- Research Centre for Medical Genetics, Moskvorechye Str. 1, Moscow 115522, Russia; (T.B.); (D.M.); (D.G.)
- Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia, Miklukho-Maklaya Str. 6, Moscow 117198, Russia
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2
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Catelli LF, Saad STO. Ex Vivo Manufacture of Megakaryocytes and Platelets from Stem Cells: Recent Advances Toward Transfusion in Humans. Stem Cells Dev 2021; 30:351-362. [PMID: 33622080 DOI: 10.1089/scd.2020.0185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The generation of ex vivo functional megakaryocytes (MK) and platelets is an important issue in transfusion medicine as donor dependence implies in limitations, such as shortage of eligible volunteers. Indeed, platelet transfusion is still a procedure that saves the lives of patients with defective platelet production. Recent technological development has enabled the isolation and expansion of stem cells that can be used as a source for the production of functional platelets for transfusion. In this review, we discuss recent approaches of in vitro or ex vivo production of MK and platelets, suggesting that, in the near future, donor-independent sources may become a possibility. The feasibility of using these cells in the clinic may be safer, and in vitro manipulation could generate universally compatible products, solving problems related to platelet refractoriness. However, functionality and survival testing of these products in human beings are scarce; therefore, additional studies are needed to consolidate this purpose.
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Affiliation(s)
- Lucas Ferioli Catelli
- Hematology and Transfusion Medicine Center, University of Campinas, Campinas, São Paulo, Brazil
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3
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Demirci S, Haro-Mora JJ, Leonard A, Drysdale C, Malide D, Keyvanfar K, Essawi K, Vizcardo R, Tamaoki N, Restifo NP, Tisdale JF, Uchida N. Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation. Stem Cell Res Ther 2020; 11:493. [PMID: 33234163 PMCID: PMC7688003 DOI: 10.1186/s13287-020-02019-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Accepted: 11/05/2020] [Indexed: 12/14/2022] Open
Abstract
Background Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders. Methods To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions. Results Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p < 0.01) and lowest number of primitive (CD34− CD235a+, 1.55%, p < 0.01) precursor cells along with the highest colony-forming units (149.8 ± 11.6, p < 0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38− CD45RA− CD49f+ CD90+) was 7.6–8.9% after 10 days of hematopoietic differentiation with 14.5% adult β-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5 ± 65.7, p < 0.001), while the CD34− CD43+ fraction produced only a small number of colonies (21.6 ± 11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center. Conclusions In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.
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Affiliation(s)
- Selami Demirci
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
| | - Juan J Haro-Mora
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
| | - Alexis Leonard
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
| | - Claire Drysdale
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
| | - Daniela Malide
- Light Microscopy Core Facility, NHLBI, NIH, Bethesda, MD, USA
| | | | - Khaled Essawi
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
| | - Raul Vizcardo
- National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA
| | - Naritaka Tamaoki
- National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA
| | - Nicholas P Restifo
- National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA
| | - John F Tisdale
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA.
| | - Naoya Uchida
- Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD, 20892, USA
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4
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Flippe L, Gaignerie A, Sérazin C, Baron O, Saulquin X, Themeli M, Guillonneau C, David L. Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells. Front Cell Dev Biol 2020; 8:577464. [PMID: 33195214 PMCID: PMC7606846 DOI: 10.3389/fcell.2020.577464] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 09/18/2020] [Indexed: 11/25/2022] Open
Abstract
Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.
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Affiliation(s)
- Léa Flippe
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Anne Gaignerie
- Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
| | - Céline Sérazin
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Olivier Baron
- Department of Pediatric Cardiac Surgery, University Hospital of Nantes, Nantes, France
| | | | - Maria Themeli
- Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Center (UMC), Amsterdam, Netherlands
| | - Carole Guillonneau
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Laurent David
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France.,Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
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5
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Bruveris FF, Ng ES, Leitoguinho AR, Motazedian A, Vlahos K, Sourris K, Mayberry R, McDonald P, Azzola L, Davidson NM, Oshlack A, Stanley EG, Elefanty AG. Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium. Development 2020; 147:dev.193037. [PMID: 33028609 PMCID: PMC7648599 DOI: 10.1242/dev.193037] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Accepted: 09/24/2020] [Indexed: 12/22/2022]
Abstract
The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17−CD34+CD43− endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17−CD34+CD43+ blood cells and SOX17+CD34+CD43− endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate. Highlighted Article: The hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like haemogenic endothelial intermediate.
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Affiliation(s)
- Freya F Bruveris
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia
| | - Elizabeth S Ng
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Ana Rita Leitoguinho
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia
| | - Ali Motazedian
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia
| | - Katerina Vlahos
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Koula Sourris
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Robyn Mayberry
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Penelope McDonald
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Lisa Azzola
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia
| | - Nadia M Davidson
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,School of BioSciences, University of Melbourne, Parkville, Victoria 3052, Australia
| | - Alicia Oshlack
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,School of BioSciences, University of Melbourne, Parkville, Victoria 3052, Australia
| | - Edouard G Stanley
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.,Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia
| | - Andrew G Elefanty
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia .,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.,Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia
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6
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A fully defined static suspension culture system for large-scale human embryonic stem cell production. Cell Death Dis 2018; 9:892. [PMID: 30166524 PMCID: PMC6117302 DOI: 10.1038/s41419-018-0863-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Revised: 06/16/2018] [Accepted: 06/21/2018] [Indexed: 12/30/2022]
Abstract
Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.
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7
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Jose SS, Tidu F, Burilova P, Kepak T, Bendickova K, Fric J. The Telomerase Complex Directly Controls Hematopoietic Stem Cell Differentiation and Senescence in an Induced Pluripotent Stem Cell Model of Telomeropathy. Front Genet 2018; 9:345. [PMID: 30210531 PMCID: PMC6123533 DOI: 10.3389/fgene.2018.00345] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Accepted: 08/09/2018] [Indexed: 01/16/2023] Open
Abstract
Telomeropathies are rare disorders associated with impaired telomere length control mechanisms that frequently result from genetic mutations in the telomerase complex. Dyskeratosis congenita is a congenital progressive telomeropathy in which mutation in the telomerase RNA component (TERC) impairs telomere maintenance leading to accelerated cellular senescence and clinical outcomes resembling premature aging. The most severe clinical feature is perturbed hematopoiesis and bone-marrow failure, but the underlying mechanisms are not fully understood. Here, we developed a model of telomerase function imbalance using shRNA to knockdown TERC expression in human induced pluripotent stem cells (iPSCs). We then promoted in vitro hematopoiesis in these cells to analyze the effects of TERC impairment. Reduced TERC expression impaired hematopoietic stem-cell (HSC) differentiation and increased the expression of cellular senescence markers and production of reactive oxygen species. Interestingly, telomere length was unaffected in shTERC knockdown iPSCs, leading to conclusion that the phenotype is controlled by non-telomeric functions of telomerase. We then assessed the effects of TERC-depletion in THP-1 myeloid cells and again observed reduced hematopoietic and myelopoietic differentiative potential. However, these cells exhibited impaired telomerase activity as verified by accelerated telomere shortening. shTERC-depleted iPSC-derived and THP-1-derived myeloid precursors had lower phagocytic capacity and increased ROS production, indicative of senescence. These findings were confirmed using a BIBR1532 TERT inhibitor, suggesting that these phenotypes are dependent on telomerase function but not directly linked to telomere length. These data provide a better understanding of the molecular processes driving the clinical signs of telomeropathies and identify novel roles of the telomerase complex other than regulating telomere length.
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Affiliation(s)
- Shyam Sushama Jose
- Cellular and Molecular Immunoregulation Group, Center for Translational Medicine, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.,Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia
| | - Federico Tidu
- Cellular and Molecular Immunoregulation Group, Center for Translational Medicine, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.,Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia
| | - Petra Burilova
- Cellular and Molecular Immunoregulation Group, Center for Translational Medicine, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.,Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia
| | - Tomas Kepak
- Pediatric Oncology Translational Research, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.,Pediatric Hematology and Oncology, The University Hospital Brno, Brno, Czechia
| | - Kamila Bendickova
- Cellular and Molecular Immunoregulation Group, Center for Translational Medicine, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia
| | - Jan Fric
- Cellular and Molecular Immunoregulation Group, Center for Translational Medicine, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia
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8
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Jose SS, Bendickova K, Fric J. High-Throughput Screening of Senescence Markers in Hematopoietic Stem Cells Derived from Induced Pluripotent Stem Cells. Methods Mol Biol 2018; 1771:121-130. [PMID: 29633209 DOI: 10.1007/978-1-4939-7792-5_10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The successful development and characterization of human induced pluripotent stem cells (iPSCs) provides a powerful tool to study the molecular mechanisms that control cell fate decisions and differentiation toward distinct lineages. Here we focus on the ability of donors derived iPSCs to differentiate toward hematopoietic progenitor cells and on the analysis of their telomere length. The ability to screen telomere length in individual donors is important for defining cellular senescence, which correlates with their differentiation potential toward hematopoietic lineages. We have modified iPSC culture protocol and telomere length analysis to suit for high throughput screening of telomere length in large number of individual donors. This approach can be used to demonstrate the heterogeneity or changes of telomere length and its shortening as an exclusion criterion for selection of suitable donors for future stem cell therapies.
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Affiliation(s)
- Shyam Sushama Jose
- Cellular and Molecular Immunoregulation Group (CMI), Center for Translational Medicine (CTM), International Clinical Research Center (ICRC), St. Anne's University Hospital Brno, Brno, Czech Republic
| | - Kamila Bendickova
- Cellular and Molecular Immunoregulation Group (CMI), Center for Translational Medicine (CTM), International Clinical Research Center (ICRC), St. Anne's University Hospital Brno, Brno, Czech Republic
| | - Jan Fric
- Cellular and Molecular Immunoregulation Group (CMI), Center for Translational Medicine (CTM), International Clinical Research Center (ICRC), St. Anne's University Hospital Brno, Brno, Czech Republic.
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9
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Rahman N, Brauer PM, Ho L, Usenko T, Tewary M, Zúñiga-Pflücker JC, Zandstra PW. Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence. Nat Commun 2017; 8:15380. [PMID: 28541275 PMCID: PMC5477512 DOI: 10.1038/ncomms15380] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2016] [Accepted: 03/22/2017] [Indexed: 12/13/2022] Open
Abstract
Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures. Intra-colony analysis revealed radial organization of CD34 and VECAD expression levels, with CD45+ blood cells emerging primarily from the colony centroid area. We identify the induced interferon gamma protein (IP-10)/p-38 MAPK signalling pathway as the mechanism for haematopoietic inhibition in our culture system. Our results highlight the role of spatial organization in hPSC-derived blood generation, and provide a quantitative platform for interrogating molecular pathways that regulate human haematopoiesis.
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Affiliation(s)
- Nafees Rahman
- Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3ES
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada M5S 3G9
| | - Patrick M. Brauer
- Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada M4N 3M5
| | - Lilian Ho
- Life Sciences (Biochemistry), University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
| | - Tatiana Usenko
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada M5S 3G9
| | - Mukul Tewary
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada M5S 3G9
- Collaborative Program in Developmental Biology, University of Toronto, Toronto, Ontario, Canada M5S 3E1
| | - Juan Carlos Zúñiga-Pflücker
- Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada M4N 3M5
- Department of Immunology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
- Medicine by Design, a Canada First Research Excellence Program at the University of Toronto, Toronto, Ontario, Canada M5S 3G9
| | - Peter W. Zandstra
- Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3ES
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada M5S 3G9
- Medicine by Design, a Canada First Research Excellence Program at the University of Toronto, Toronto, Ontario, Canada M5S 3G9
- Terrence Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1
- Collaborative Program in Developmental Biology, University of Toronto, Toronto, Ontario, Canada M5S 3E1
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10
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Paes BCMF, Moço PD, Pereira CG, Porto GS, de Sousa Russo EM, Reis LCJ, Covas DT, Picanço-Castro V. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation. Cell Biol Toxicol 2016; 33:233-250. [PMID: 28039590 DOI: 10.1007/s10565-016-9377-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2016] [Accepted: 12/18/2016] [Indexed: 01/19/2023]
Abstract
Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.
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Affiliation(s)
- Bárbara Cristina Martins Fernandes Paes
- Ribeirão Preto Medical School and Center for Cell-based Therapy (CTC), University of São Paulo, São Paulo, Brazil
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil
| | - Pablo Diego Moço
- Ribeirão Preto Medical School and Center for Cell-based Therapy (CTC), University of São Paulo, São Paulo, Brazil
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil
| | - Cristiano Gonçalves Pereira
- School of Economics, Business Administration and Accounting at Ribeirão Preto, University of São Paulo, São Paulo, Brazil
| | - Geciane Silveira Porto
- School of Economics, Business Administration and Accounting at Ribeirão Preto, University of São Paulo, São Paulo, Brazil
| | - Elisa Maria de Sousa Russo
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil
- Ribeirão Preto Pharmaceutical Sciences School, University of São Paulo, São Paulo, Brazil
| | - Luiza Cunha Junqueira Reis
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil
- Ribeirão Preto Pharmaceutical Sciences School, University of São Paulo, São Paulo, Brazil
| | - Dimas Tadeu Covas
- Ribeirão Preto Medical School and Center for Cell-based Therapy (CTC), University of São Paulo, São Paulo, Brazil
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil
| | - Virginia Picanço-Castro
- Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, São Paulo, 14051-140, Brazil.
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11
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Murray A, Letourneau A, Canzonetta C, Stathaki E, Gimelli S, Sloan-Bena F, Abrehart R, Goh P, Lim S, Baldo C, Dagna-Bricarelli F, Hannan S, Mortensen M, Ballard D, Syndercombe Court D, Fusaki N, Hasegawa M, Smart TG, Bishop C, Antonarakis SE, Groet J, Nizetic D. Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration. Stem Cells 2016; 33:2077-84. [PMID: 25694335 PMCID: PMC4737213 DOI: 10.1002/stem.1968] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2014] [Accepted: 01/17/2015] [Indexed: 01/11/2023]
Abstract
Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.
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Affiliation(s)
- Aoife Murray
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom.,The LonDownS Consortium, Wellcome Trust, London, United Kingdom
| | - Audrey Letourneau
- Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland
| | - Claudia Canzonetta
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom
| | - Elisavet Stathaki
- Service of Genetic Medicine, University Geneva Hospitals, Geneva, Switzerland
| | - Stefania Gimelli
- Service of Genetic Medicine, University Geneva Hospitals, Geneva, Switzerland
| | | | - Robert Abrehart
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom
| | - Pollyanna Goh
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom.,The LonDownS Consortium, Wellcome Trust, London, United Kingdom
| | - Shuhui Lim
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
| | - Chiara Baldo
- Human Genetics Laboratory, Galliera Hospital, Genoa, Italy
| | | | - Saad Hannan
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom
| | - Martin Mortensen
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom
| | - David Ballard
- Department of Forensic and Analytical Science, King's College, London, United Kingdom
| | | | - Noemi Fusaki
- Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan
| | | | - Trevor G Smart
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom
| | - Cleo Bishop
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom
| | - Stylianos E Antonarakis
- Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland
| | - Jürgen Groet
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom.,Stem Cell Laboratory, National Centre for Bowel Research and Surgical Innovation, Queen Mary University of London, London, United Kingdom.,The LonDownS Consortium, Wellcome Trust, London, United Kingdom
| | - Dean Nizetic
- The Blizard Institute, Barts and The London School of Medicine, London, United Kingdom.,The LonDownS Consortium, Wellcome Trust, London, United Kingdom.,Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
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12
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Asprer JST, Lakshmipathy U. Current methods and challenges in the comprehensive characterization of human pluripotent stem cells. Stem Cell Rev Rep 2016; 11:357-72. [PMID: 25504379 DOI: 10.1007/s12015-014-9580-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Pluripotent stem cells (PSCs) are powerful tools for basic scientific research and promising agents for drug discovery and regenerative medicine. Technological advances have made it increasingly easy to generate PSCs but the various lines generated may differ in their characteristics based on their origin, derivation, number of passages, and culture conditions. In order to confirm the pluripotency, quality, identity, and safety of pluripotent cell lines as they are derived and maintained, it is critical to perform a panel of characterization assays. Functional pluripotency is determined using tests that rely on the expression of specific markers in the undifferentiated and differentiated states; tests for quality, identity and safety are less specialized. This article provides a comprehensive review of current practices in PSC characterization and explores challenges in the field, from the selection of markers to the development of simple and scalable methods. It also delves into emerging trends like the adoption of alternative assays that could be used to supplement or replace traditional methods, specifically the use of in silico assays for determining pluripotency.
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Affiliation(s)
- Joanna S T Asprer
- Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA, 92008, USA
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13
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Souza GTD, Maranduba CP, Souza CMD, Amaral DLASD, Guia FCD, Zanette RDSS, Rettore JVP, Rabelo NC, Nascimento LM, Pinto &IFN, Farani JB, Neto AEH, Silva FDS, Maranduba CMDC, Atalla A. Advances in cellular technology in the hematology field: What have we learned so far? World J Stem Cells 2015; 7:106-115. [PMID: 25621110 PMCID: PMC4300920 DOI: 10.4252/wjsc.v7.i1.106] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 09/12/2014] [Accepted: 09/19/2014] [Indexed: 02/07/2023] Open
Abstract
Despite the advances in the hematology field, blood transfusion-related iatrogenesis is still a major issue to be considered during such procedures due to blood antigenic incompatibility. This places pluripotent stem cells as a possible ally in the production of more suitable blood products. The present review article aims to provide a comprehensive summary of the state-of-the-art concerning the differentiation of both embryonic stem cells and induced pluripotent stem cells to hematopoietic cell lines. Here, we review the most recently published protocols to achieve the production of blood cells for future application in hemotherapy, cancer therapy and basic research.
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14
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Hermanson DL, Ni Z, Kaufman DS. Human Pluripotent Stem Cells as a Renewable Source of Natural Killer Cells. SPRINGERBRIEFS IN STEM CELLS 2015. [DOI: 10.1007/978-94-017-7312-6_5] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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15
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Yuan X, Braunstein EM, Ye Z, Liu CF, Chen G, Zou J, Cheng L, Brodsky RA. Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells. Stem Cells Transl Med 2013; 2:819-29. [PMID: 24113066 DOI: 10.5966/sctm.2013-0069] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
PIG-A is an X-linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG-A mutant cells have a deficiency or absence of all GPI-anchored proteins (GPI-APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG-A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG-A gene targeting and an inducible PIG-A expression system, we have established, for the first time, a conditional PIG-A knockout model in human iPSCs that allows for the production of GPI-AP-deficient blood cells. PIG-A-null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG-A-null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG-A transgene expression allowed for the generation of GPI-AP-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development.
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Affiliation(s)
- Xuan Yuan
- Division of Hematology, Department of Medicine, School of Medicine, and
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16
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Bock AM, Knorr D, Kaufman DS. Development, expansion, and in vivo monitoring of human NK cells from human embryonic stem cells (hESCs) and and induced pluripotent stem cells (iPSCs). J Vis Exp 2013:e50337. [PMID: 23644738 DOI: 10.3791/50337] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.
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Affiliation(s)
- Allison M Bock
- Department of Medicine Hematology, Oncology, and Transplant, University of Minnesota, Minneapolis, USA
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17
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Hotta R, Stamp LA, Foong JPP, McConnell SN, Bergner AJ, Anderson RB, Enomoto H, Newgreen DF, Obermayr F, Furness JB, Young HM. Transplanted progenitors generate functional enteric neurons in the postnatal colon. J Clin Invest 2013; 123:1182-91. [PMID: 23454768 DOI: 10.1172/jci65963] [Citation(s) in RCA: 124] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2012] [Accepted: 12/11/2012] [Indexed: 01/11/2023] Open
Abstract
Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.
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Affiliation(s)
- Ryo Hotta
- Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Victoria, Australia
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18
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Ni Z, Knorr DA, Kaufman DS. Hematopoietic and nature killer cell development from human pluripotent stem cells. Methods Mol Biol 2013; 1029:33-41. [PMID: 23756940 DOI: 10.1007/978-1-62703-478-4_3] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Natural killer (NK) cells are key effectors of the innate immune system, protecting the host from a variety of infections, as well as malignant cells. Recent advances in the field of NK cell biology have led to a better understanding of how NK cells develop. This progress has directly translated to improved outcomes in patients receiving hematopoietic stem cell transplants to treat potentially lethal malignancies. However, key differences between mouse and human NK cell development and biology limits the use of rodents to attain a more in depth understanding of NK cell development. Therefore, a readily accessible and genetically tractable cell source to study human NK cell development is warranted. Our lab has pioneered the development of lymphocytes, specifically NK cells, from human embryonic stem cells (hESCs) and more recently induced pluripotent stem cells (iPSCs). This chapter describes a reliable method to generate NK cells from hESCs and iPSCs using murine stromal cell lines. Additionally, we include an updated approach using a spin-embryoid body (spin-EB) differentiation system that allows for human NK cell development completely defined in vitro conditions.
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Affiliation(s)
- Zhenya Ni
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
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19
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Prowse ABJ, Chong F, Elliott DA, Elefanty AG, Stanley EG, Gray PP, Munro TP, Osborne GW. Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro. PLoS One 2012; 7:e52214. [PMID: 23284940 PMCID: PMC3526579 DOI: 10.1371/journal.pone.0052214] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2012] [Accepted: 11/09/2012] [Indexed: 01/07/2023] Open
Abstract
Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.
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Affiliation(s)
- Andrew B J Prowse
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Australia.
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20
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Outten JT, Gadue P, French DL, Diamond SL. High-throughput screening assay for embryoid body differentiation of human embryonic stem cells. CURRENT PROTOCOLS IN STEM CELL BIOLOGY 2012; Chapter 1:Unit 1D.6. [PMID: 22415836 DOI: 10.1002/9780470151808.sc01d06s20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Serum-free human pluripotent stem cell media offer the potential to develop reproducible clinically applicable differentiation strategies and protocols. The vast array of possible growth factor and cytokine combinations for media formulations makes differentiation protocol optimization both labor and cost-intensive. This unit describes a 96-well plate, 4-color flow cytometry-based screening assay to optimize pluripotent stem cell differentiation protocols. We provide conditions both to differentiate human embryonic stem cells (hESCs) into the three primary germ layers, ectoderm, endoderm, and mesoderm, and to utilize flow cytometry to distinguish between them. This assay exhibits low inter-well variability and can be utilized to efficiently screen a variety of media formulations, reducing cost, incubator space, and labor. Protocols can be adapted to a variety of differentiation stages and lineages.
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Affiliation(s)
- Joel T Outten
- Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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21
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Goulburn AL, Stanley EG, Elefanty AG, Anderson SA. Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells. Stem Cell Res 2011; 8:416-26. [PMID: 22280980 DOI: 10.1016/j.scr.2011.12.002] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2011] [Revised: 11/29/2011] [Accepted: 12/03/2011] [Indexed: 12/15/2022] Open
Affiliation(s)
- Adam L Goulburn
- Department of Psychiatry, Weill Cornell Medical College, New York, NY 10065, USA.
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22
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Yung S, Ledran M, Moreno-Gimeno I, Conesa A, Montaner D, Dopazo J, Dimmick I, Slater NJ, Marenah L, Real PJ, Paraskevopoulou I, Bisbal V, Burks D, Santibanez-Koref M, Moreno R, Mountford J, Menendez P, Armstrong L, Lako M. Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells. Hum Mol Genet 2011; 20:4932-46. [PMID: 21937587 DOI: 10.1093/hmg/ddr431] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.
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Affiliation(s)
- Sun Yung
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK
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23
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Kim JM, Moon SH, Lee SG, Cho YJ, Hong KS, Lee JH, Lee HJ, Chung HM. Assessment of differentiation aspects by the morphological classification of embryoid bodies derived from human embryonic stem cells. Stem Cells Dev 2011; 20:1925-35. [PMID: 21388292 DOI: 10.1089/scd.2010.0476] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.
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Affiliation(s)
- Jung Mo Kim
- Stem Cell Research Lab, CHA Stem Cell Institute, CHA University, Seoul, Republic of Korea
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24
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Hexum MK, Tian X, Kaufman DS. In vivo evaluation of putative hematopoietic stem cells derived from human pluripotent stem cells. Methods Mol Biol 2011; 767:433-47. [PMID: 21822894 DOI: 10.1007/978-1-61779-201-4_32] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Efficient derivation and isolation of hematopoietic stem cells (HSCs) from human pluripotent stem cell (hPSC) populations remains a major goal in the field of developmental hematopoiesis. These enticing pluripotent stem cells (comprising both human embryonic stem cells and induced pluripotent stem cells) have been successfully used to generate a wide array of hematopoietic cells in vitro, from primitive hematoendothelial precursors to mature myeloid, erythroid, and lymphoid lineage cells. However, to date, PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo - the test by which putative HSCs are defined. Successful generation and characterization of HSCs from hPSCs not only requires an efficient in vitro differentiation system that provides insight into the developmental fate of hPSC-derived cells, but also necessitates an in vivo engraftment model that allows identification of specific mechanisms that hinder or promote hematopoietic engraftment. In this chapter, we will describe a method that utilizes firefly luciferase-expressing hPSCs and bioluminescent imaging to noninvasively track the survival, proliferation, and migration of transplanted hPSC-derived cells. Combined with lineage and functional analyses of engrafted cells, this system is a useful tool to gain insight into the in vivo potential of hematopoietic cells generated from hPSCs.
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Affiliation(s)
- Melinda K Hexum
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
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25
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Moreno-Gimeno I, Ledran MH, Lako M. Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. Invited review following the FEBS Anniversary Prize received on 5 July 2009 at the 34th FEBS Congress in Prague. FEBS J 2010; 277:5014-25. [PMID: 21087454 DOI: 10.1111/j.1742-4658.2010.07926.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Human embryonic stem cells (hESCs) and induced pluripotent stem cells are excellent models for the study of embryonic hematopoiesis in vitro, aiding the design of new differentiation models that may be applicable to cell-replacement therapies. Adult and fetal hematopoietic stem cells are currently being used in biomedical applications; however, the latest advances in regenerative medicine and stem cell biology suggest that hESC-derived hematopoietic stem cells are an outstanding tool for enhancing immunotherapy and treatments for blood disorders and cancer, for example. In this review, we compare various methods used for inducing in vitro hematopoietic differentiation from hESCs, based on co-culture with stromal cells or formation of embryoid bodies, and analyse their ability to give rise to hematopoietic precursors, with emphasis on their engraftment potential as a measure of their functionality in vivo.
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26
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Prowse ABJ, Doran MR, Cooper-White JJ, Chong F, Munro TP, Fitzpatrick J, Chung TL, Haylock DN, Gray PP, Wolvetang EJ. Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media. Biomaterials 2010; 31:8281-8. [PMID: 20674971 DOI: 10.1016/j.biomaterials.2010.07.037] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2010] [Accepted: 07/07/2010] [Indexed: 01/22/2023]
Abstract
Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.
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Affiliation(s)
- Andrew B J Prowse
- The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
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27
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Fourier transform infrared microspectroscopy identifies early lineage commitment in differentiating human embryonic stem cells. Stem Cell Res 2010; 4:140-7. [DOI: 10.1016/j.scr.2009.11.002] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/20/2009] [Accepted: 11/25/2009] [Indexed: 11/19/2022] Open
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Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases. Proc Natl Acad Sci U S A 2009; 106:9826-30. [PMID: 19482945 DOI: 10.1073/pnas.0904689106] [Citation(s) in RCA: 168] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The innovation of reprogramming somatic cells to induced pluripotent stem cells provides a possible new approach to treat beta-thalassemia and other genetic diseases such as sickle cell anemia. Induced pluripotent stem (iPS) cells can be made from these patients' somatic cells and the mutation in the beta-globin gene corrected by gene targeting, and the cells differentiated into hematopoietic cells to be returned to the patient. In this study, we reprogrammed the skin fibroblasts of a patient with homozygous beta(0) thalassemia into iPS cells, and showed that the iPS cells could be differentiated into hematopoietic cells that synthesized hemoglobin. Prenatal diagnosis and selective abortion have been effective in decreasing the number of beta-thalassemia births in some countries that have instituted carrier screening and genetic counseling. To make use of the cells from the amniotic fluid or chorionic villus sampling that are used for prenatal diagnosis, we also showed that these cells could be reprogrammed into iPS cells. This raises the possibility of providing a new option following prenatal diagnosis of a fetus affected by a severe illness. Currently, the parents would choose either to terminate the pregnancy or continue it and take care of the sick child after birth. The cells for prenatal diagnosis can be converted into iPS cells for treatment in the perinatal periods. Early treatment has the advantage of requiring much fewer cells than adult treatment, and can also prevent organ damage in those diseases in which damage can begin in utero or at an early age.
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