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Filippi L, Innocenti F, Pascarella F, Scaramuzzo RT, Morganti R, Bagnoli P, Cammalleri M, Dal Monte M, Calvani M, Pini A. β 3-Adrenoceptor Agonism to Mimic the Biological Effects of Intrauterine Hypoxia: Taking Great Strides Toward a Pharmacological Artificial Placenta. Med Res Rev 2025; 45:842-866. [PMID: 39604126 PMCID: PMC11976384 DOI: 10.1002/med.22092] [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: 07/31/2024] [Revised: 10/24/2024] [Accepted: 11/11/2024] [Indexed: 11/29/2024]
Abstract
At different stages of life, from embryonic to postnatal, varying oxygen concentrations modulate cellular gene expression by enhancing or repressing hypoxia-inducible transcription factors. During embryonic/fetal life, these genes encode proteins involved in adapting to a low-oxygen environment, including the induction of specific enzymes related to glycolytic metabolism, erythropoiesis, angiogenesis, and vasculogenesis. However, oxygen concentrations fluctuate during intrauterine life, enabling the induction of tissue-specific differentiation processes. Fetal well-being is thus closely linked to the physiological benefits of a dynamically hypoxic environment. Premature birth entails the precocious exposure of the immature fetus to a more oxygen-rich environment compared to the womb. As a result, preterm newborns face a condition of relative hyperoxia, which alters the postnatal development of organs and contributes to prematurity-related diseases. However, until recently, the molecular mechanism by which high oxygen tension alters normal fetal differentiation remained unclear. In this review, we discuss the research trajectory followed by our research group, which suggests that early exposure to a relatively hyperoxic environment may impair preterm neonates due to reduced expression of the β3-adrenoceptor. Additionally, we explore how these impairments could be prevented through the pharmacological stimulation of the remaining β3-adrenoceptors. Recent preclinical studies demonstrate that pharmacological stimulation of the β3-adrenoceptor can decouple exposure to hyperoxia from its harmful effects, offering a glimpse of the possibility to recreating the conditions typical of intrauterine life, even after premature birth.
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Affiliation(s)
- Luca Filippi
- Neonatology UnitAzienda Ospedaliero‐Universitaria PisanaPisaItaly
- Department of Clinical and Experimental MedicineUniversity of PisaPisaItaly
| | | | | | | | - Riccardo Morganti
- Section of StatisticsAzienda Ospedaliero‐Universitaria PisanaPisaItaly
| | - Paola Bagnoli
- Department of Biology, Unit of General PhysiologyUniversity of PisaPisaItaly
| | - Maurizio Cammalleri
- Department of Biology, Unit of General PhysiologyUniversity of PisaPisaItaly
| | - Massimo Dal Monte
- Department of Biology, Unit of General PhysiologyUniversity of PisaPisaItaly
| | - Maura Calvani
- Department of Pediatric Hematology‐OncologyMeyer Children's Hospital IRCCSFlorenceItaly
| | - Alessandro Pini
- Department of Experimental and Clinical MedicineUniversity of FlorenceFlorenceItaly
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2
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Grattoni A, Korbutt G, Tomei AA, García AJ, Pepper AR, Stabler C, Brehm M, Papas K, Citro A, Shirwan H, Millman JR, Melero-Martin J, Graham M, Sefton M, Ma M, Kenyon N, Veiseh O, Desai TA, Nostro MC, Marinac M, Sykes M, Russ HA, Odorico J, Tang Q, Ricordi C, Latres E, Mamrak NE, Giraldo J, Poznansky MC, de Vos P. Harnessing cellular therapeutics for type 1 diabetes mellitus: progress, challenges, and the road ahead. Nat Rev Endocrinol 2025; 21:14-30. [PMID: 39227741 PMCID: PMC11938328 DOI: 10.1038/s41574-024-01029-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/06/2024] [Indexed: 09/05/2024]
Abstract
Type 1 diabetes mellitus (T1DM) is a growing global health concern that affects approximately 8.5 million individuals worldwide. T1DM is characterized by an autoimmune destruction of pancreatic β cells, leading to a disruption in glucose homeostasis. Therapeutic intervention for T1DM requires a complex regimen of glycaemic monitoring and the administration of exogenous insulin to regulate blood glucose levels. Advances in continuous glucose monitoring and algorithm-driven insulin delivery devices have improved the quality of life of patients. Despite this, mimicking islet function and complex physiological feedback remains challenging. Pancreatic islet transplantation represents a potential functional cure for T1DM but is hindered by donor scarcity, variability in harvested cells, aggressive immunosuppressive regimens and suboptimal clinical outcomes. Current research is directed towards generating alternative cell sources, improving transplantation methods, and enhancing cell survival without chronic immunosuppression. This Review maps the progress in cell replacement therapies for T1DM and outlines the remaining challenges and future directions. We explore the state-of-the-art strategies for generating replenishable β cells, cell delivery technologies and local targeted immune modulation. Finally, we highlight relevant animal models and the regulatory aspects for advancing these technologies towards clinical deployment.
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Affiliation(s)
- Alessandro Grattoni
- Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX, USA.
- Department of Surgery, Houston Methodist Hospital, Houston, TX, USA.
- Department of Radiation Oncology, Houston Methodist Hospital, Houston, TX, USA.
| | - Gregory Korbutt
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
| | - Alice A Tomei
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA
- Department of Biomedical Engineering, University of Miami, Miami, FL, USA
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
- Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Andrés J García
- Woodruff School of Mechanical Engineering and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA
| | - Andrew R Pepper
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
| | - Cherie Stabler
- J. Crayton Pruitt Family Department of Biomedical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, USA
- Diabetes Institute, University of Florida, Gainesville, FL, USA
| | - Michael Brehm
- Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Klearchos Papas
- Department of Surgery, The University of Arizona, Tucson, AZ, USA
| | - Antonio Citro
- Diabetes Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy
| | - Haval Shirwan
- Department of Pediatrics, Ellis Fischel Cancer Center, School of Medicine, University of Missouri, Columbia, MO, USA
| | - Jeffrey R Millman
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
| | - Juan Melero-Martin
- Department of Cardiac Surgery, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Harvard Stem Cell Institute, Cambridge, MA, USA
| | - Melanie Graham
- Department of Surgery, University of Minnesota, Minneapolis, MN, USA
- Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN, USA
| | - Michael Sefton
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
- Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
| | - Minglin Ma
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA
| | - Norma Kenyon
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Omid Veiseh
- Department of Bioengineering, Rice University, Houston, TX, USA
| | - Tejal A Desai
- University of California, San Francisco, Department of Bioengineering and Therapeutic Sciences, San Francisco, CA, USA
- Brown University, School of Engineering, Providence, RI, USA
| | - M Cristina Nostro
- McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada
- Department of Physiology, University of Toronto, Toronto, ON, Canada
| | | | - Megan Sykes
- Department of Medicine, Columbia Center for Translational Immunology, Columbia University, New York, NY, USA
- Department of Microbiology and Immunology, Columbia University, New York, NY, USA
- Department of Surgery, Columbia University, New York, NY, USA
| | - Holger A Russ
- Diabetes Institute, University of Florida, Gainesville, FL, USA
- Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA
| | - Jon Odorico
- UW Health Transplant Center, Madison, WI, USA
- Division of Transplantation, Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
| | - Qizhi Tang
- Diabetes Center, University of California San Francisco, San Francisco, CA, USA
- Department of Surgery, University of California San Francisco, San Francisco, CA, US
- Gladstone Institute of Genomic Immunology, University of California San Francisco, San Francisco, CA, USA
| | - Camillo Ricordi
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Esther Latres
- Research Department, Breakthrough T1D, New York, NY, USA
| | | | - Jaime Giraldo
- Research Department, Breakthrough T1D, New York, NY, USA.
| | - Mark C Poznansky
- Vaccine and Immunotherapy Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
| | - Paul de Vos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Groningen, Netherlands.
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3
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Hashemi M, Finklea FB, Hammons H, Tian Y, Young N, Kim E, Halloin C, Triebert W, Zweigerdt R, Mitra AK, Lipke EA. Hydrogel microsphere stem cell encapsulation enhances cardiomyocyte differentiation and functionality in scalable suspension system. Bioact Mater 2025; 43:423-440. [PMID: 39399838 PMCID: PMC11471139 DOI: 10.1016/j.bioactmat.2024.08.043] [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: 04/18/2024] [Revised: 08/30/2024] [Accepted: 08/31/2024] [Indexed: 10/15/2024] Open
Abstract
A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.
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Affiliation(s)
| | - Ferdous B. Finklea
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
| | - Hanna Hammons
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
| | - Yuan Tian
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
| | - Nathan Young
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
| | - Emma Kim
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
| | - Caroline Halloin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hanover, Germany
| | - Wiebke Triebert
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hanover, Germany
| | - Robert Zweigerdt
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hanover, Germany
| | - Amit Kumar Mitra
- Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, Auburn, AL, United States
| | - Elizabeth A. Lipke
- Department of Chemical Engineering, Auburn University, Auburn, AL, United States
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4
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Mensah IK, Gowher H. Signaling Pathways Governing Cardiomyocyte Differentiation. Genes (Basel) 2024; 15:798. [PMID: 38927734 PMCID: PMC11202427 DOI: 10.3390/genes15060798] [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: 05/24/2024] [Revised: 06/13/2024] [Accepted: 06/13/2024] [Indexed: 06/28/2024] Open
Abstract
Cardiomyocytes are the largest cell type that make up the heart and confer beating activity to the heart. The proper differentiation of cardiomyocytes relies on the efficient transmission and perception of differentiation cues from several signaling pathways that influence cardiomyocyte-specific gene expression programs. Signaling pathways also mediate intercellular communications to promote proper cardiomyocyte differentiation. We have reviewed the major signaling pathways involved in cardiomyocyte differentiation, including the BMP, Notch, sonic hedgehog, Hippo, and Wnt signaling pathways. Additionally, we highlight the differences between different cardiomyocyte cell lines and the use of these signaling pathways in the differentiation of cardiomyocytes from stem cells. Finally, we conclude by discussing open questions and current gaps in knowledge about the in vitro differentiation of cardiomyocytes and propose new avenues of research to fill those gaps.
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Affiliation(s)
| | - Humaira Gowher
- Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
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5
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Hogrebe NJ, Ishahak M, Millman JR. Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes. Cell Stem Cell 2023; 30:530-548. [PMID: 37146579 PMCID: PMC10167558 DOI: 10.1016/j.stem.2023.04.002] [Citation(s) in RCA: 69] [Impact Index Per Article: 34.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 03/20/2023] [Accepted: 04/05/2023] [Indexed: 05/07/2023]
Abstract
The generation of islet-like endocrine clusters from human pluripotent stem cells (hPSCs) has the potential to provide an unlimited source of insulin-producing β cells for the treatment of diabetes. In order for this cell therapy to become widely adopted, highly functional and well-characterized stem cell-derived islets (SC-islets) need to be manufactured at scale. Furthermore, successful SC-islet replacement strategies should prevent significant cell loss immediately following transplantation and avoid long-term immune rejection. This review highlights the most recent advances in the generation and characterization of highly functional SC-islets as well as strategies to ensure graft viability and safety after transplantation.
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Affiliation(s)
- Nathaniel J Hogrebe
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63130, USA.
| | - Matthew Ishahak
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63130, USA
| | - Jeffrey R Millman
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63130, USA; Department of Biomedical Engineering, Washington University in St. Louis, 1 Brookings Drive, St. Louis, MO 63130, USA.
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6
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Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment. Int J Mol Sci 2022; 23:ijms232415785. [PMID: 36555427 PMCID: PMC9779514 DOI: 10.3390/ijms232415785] [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: 10/23/2022] [Revised: 12/06/2022] [Accepted: 12/07/2022] [Indexed: 12/14/2022] Open
Abstract
Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.
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Ho DLL, Lee S, Du J, Weiss JD, Tam T, Sinha S, Klinger D, Devine S, Hamfeldt A, Leng HT, Herrmann JE, He M, Fradkin LG, Tan TK, Standish D, Tomasello P, Traul D, Dianat N, Ladi R, Vicard Q, Katikireddy K, Skylar‐Scott MA. Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors. Adv Healthc Mater 2022; 11:e2201138. [PMID: 36314397 PMCID: PMC10234214 DOI: 10.1002/adhm.202201138] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 10/10/2022] [Indexed: 01/28/2023]
Abstract
Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.
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Affiliation(s)
- Debbie L. L. Ho
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Stacey Lee
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Jianyi Du
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | | | - Tony Tam
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Soham Sinha
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Danielle Klinger
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Sean Devine
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Art Hamfeldt
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Hope T. Leng
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Jessica E. Herrmann
- Department of BioengineeringStanford UniversityStanfordCA94305USA
- School of MedicineStanford UniversityStanfordCA94305USA
| | - Mengdi He
- Materials Science and EngineeringStanford UniversityStanfordCA94305USA
| | - Lee G. Fradkin
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Tze Kai Tan
- Institute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanfordCA94305USA
- Department of GeneticsStanford University School of MedicineStanfordCA94305USA
- Department of PathologyStanford University School of MedicineStanfordCA94305USA
| | - David Standish
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Peter Tomasello
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Donald Traul
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Noushin Dianat
- Sartorius Stedim France S.A.SZone Industrielle les PaludsAvenue de Jouques CS 71058Aubagne Cedex13781France
| | - Rukmini Ladi
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Quentin Vicard
- Sartorius Stedim France S.A.SZone Industrielle les PaludsAvenue de Jouques CS 71058Aubagne Cedex13781France
| | | | - Mark A. Skylar‐Scott
- Department of BioengineeringStanford UniversityStanfordCA94305USA
- Basic Science and Engineering InitiativeChildren's Heart CenterStanford UniversityStanfordCA94305USA
- Chan Zuckerberg BiohubSan FranciscoCA94158USA
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Khadim RR, Vadivelu RK, Utami T, Torizal FG, Nishikawa M, Sakai Y. Integrating Oxygen and 3D Cell Culture System: A Simple Tool to Elucidate the Cell Fate Decision of hiPSCs. Int J Mol Sci 2022; 23:ijms23137272. [PMID: 35806277 PMCID: PMC9266965 DOI: 10.3390/ijms23137272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Revised: 06/28/2022] [Accepted: 06/29/2022] [Indexed: 11/23/2022] Open
Abstract
Oxygen, as an external environmental factor, plays a role in the early differentiation of human stem cells, such as induced pluripotent stem cells (hiPSCs). However, the effect of oxygen concentration on the early-stage differentiation of hiPSC is not fully understood, especially in 3D aggregate cultures. In this study, we cultivated the 3D aggregation of hiPSCs on oxygen-permeable microwells under different oxygen concentrations ranging from 2.5 to 20% and found that the aggregates became larger, corresponding to the increase in oxygen level. In a low oxygen environment, the glycolytic pathway was more profound, and the differentiation markers of the three germ layers were upregulated, suggesting that the oxygen concentration can function as a regulator of differentiation during the early stage of development. In conclusion, culturing stem cells on oxygen-permeable microwells may serve as a platform to investigate the effect of oxygen concentration on diverse cell fate decisions during development.
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Affiliation(s)
- Rubina Rahaman Khadim
- Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan; (T.U.); (F.G.T.); (Y.S.)
- Correspondence: (R.R.K.); (R.K.V.)
| | - Raja Kumar Vadivelu
- Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan;
- Human Biomimetic System, RIKEN Hakubi Research Team, RIKEN Cluster for Pioneering Research (CPR), Wako 351-0198, Saitama, Japan
- Correspondence: (R.R.K.); (R.K.V.)
| | - Tia Utami
- Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan; (T.U.); (F.G.T.); (Y.S.)
| | - Fuad Gandhi Torizal
- Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan; (T.U.); (F.G.T.); (Y.S.)
| | - Masaki Nishikawa
- Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan;
| | - Yasuyuki Sakai
- Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan; (T.U.); (F.G.T.); (Y.S.)
- Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8654, Japan;
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9
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Cha JM, Hwang YS, Kang DK, Lee J, Cooper ES, Mantalaris A. Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs. Tissue Eng Regen Med 2022; 19:739-754. [PMID: 35532736 PMCID: PMC9294093 DOI: 10.1007/s13770-022-00447-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2022] [Revised: 02/06/2022] [Accepted: 02/20/2022] [Indexed: 10/18/2022] Open
Abstract
BACKGROUND As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. METHODS An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. RESULTS Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. CONCLUSION This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.
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10
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Decarli MC, Mizukami A, Azoubel RA, Neto PI, Mota C, Moraes ÂM, Silva JVL, Moroni L. Static systems to obtain 3D spheroid cell models: a cost analysis comparing the implementation of four types of microwell array inserts. Biochem Eng J 2022. [DOI: 10.1016/j.bej.2022.108414] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
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11
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Francis HS, Harold CL, Beagrie RA, King AJ, Gosden ME, Blayney JW, Jeziorska DM, Babbs C, Higgs DR, Kassouf MT. Scalable in vitro production of defined mouse erythroblasts. PLoS One 2022; 17:e0261950. [PMID: 34995303 PMCID: PMC8741028 DOI: 10.1371/journal.pone.0261950] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Accepted: 12/14/2021] [Indexed: 01/23/2023] Open
Abstract
Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.
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Affiliation(s)
- Helena S. Francis
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Caroline L. Harold
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Robert A. Beagrie
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Andrew J. King
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Matthew E. Gosden
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Joseph W. Blayney
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Danuta M. Jeziorska
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Christian Babbs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Douglas R. Higgs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Mira T. Kassouf
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
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12
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Oxygen as a Master Regulator of Human Pluripotent Stem Cell Function and Metabolism. J Pers Med 2021; 11:jpm11090905. [PMID: 34575682 PMCID: PMC8466012 DOI: 10.3390/jpm11090905] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 08/30/2021] [Accepted: 09/08/2021] [Indexed: 12/11/2022] Open
Abstract
Human-induced pluripotent stem cells (hiPSCs) offer numerous possibilities in science and medicine, particularly when combined with precise genome editing methods. hiPSCs are artificially generated equivalents of human embryonic stem cells (hESCs), which possess an unlimited ability to self-renew and the potential to differentiate into any cell type of the human body. Importantly, generating patient-specific hiPSCs enables personalized drug testing or autologous cell therapy upon differentiation into a desired cell line. However, to ensure the highest standard of hiPSC-based biomedical products, their safety and reliability need to be proved. One of the key factors influencing human pluripotent stem cell (hPSC) characteristics and function is oxygen concentration in their microenvironment. In recent years, emerging data have pointed toward the beneficial effect of low oxygen pressure (hypoxia) on both hiPSCs and hESCs. In this review, we examine the state-of-the-art research on the oxygen impact on hiPSC functions and activity with an emphasis on their niche, metabolic state, reprogramming efficiency, and differentiation potential. We also discuss the similarities and differences between PSCs and cancer stem cells (CSCs) with respect to the role of oxygen in both cell types.
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13
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Response of Pluripotent Stem Cells to Environmental Stress and Its Application for Directed Differentiation. BIOLOGY 2021; 10:biology10020084. [PMID: 33498611 PMCID: PMC7912122 DOI: 10.3390/biology10020084] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 01/14/2021] [Accepted: 01/20/2021] [Indexed: 12/15/2022]
Abstract
Simple Summary Environmental changes in oxygen concentration, temperature, and mechanical stimulation lead to the activation of specific transcriptional factors and induce the expression of each downstream gene. In general, these responses are protective machinery against such environmental stresses, while these transcriptional factors also regulate cell proliferation, differentiation, and organ development in mammals. In the case of pluripotent stem cells, similar response mechanisms normally work and sometimes stimulate the differentiation cues. Up to now, differentiation protocols utilizing such environmental stresses have been reported to obtain various types of somatic cells from pluripotent stem cells. Basically, environmental stresses as hypoxia (low oxygen), hyperoxia, (high oxygen) and mechanical stress from cell culture plates are relatively safer than chemicals and gene transfers, which affect the genome irreversibly. Therefore, protocols designed with such environments in mind could be useful for the technology development of cell therapy and regenerative medicine. In this manuscript, we summarize recent findings of environmental stress-induced differentiation protocols and discuss their mechanisms. Abstract Pluripotent stem cells have unique characteristics compared to somatic cells. In this review, we summarize the response to environmental stresses (hypoxic, oxidative, thermal, and mechanical stresses) in embryonic stem cells (ESCs) and their applications in the differentiation methods directed to specific lineages. Those stresses lead to activation of each specific transcription factor followed by the induction of downstream genes, and one of them regulates lineage specification. In short, hypoxic stress promotes the differentiation of ESCs to mesodermal lineages via HIF-1α activation. Concerning mechanical stress, high stiffness tends to promote mesodermal differentiation, while low stiffness promotes ectodermal differentiation via the modulation of YAP1. Furthermore, each step in the same lineage differentiation favors each appropriate stiffness of culture plate; for example, definitive endoderm favors high stiffness, while pancreatic progenitor favors low stiffness during pancreatic differentiation of human ESCs. Overall, treatments utilizing those stresses have no genotoxic or carcinogenic effects except oxidative stress; therefore, the differentiated cells are safe and could be useful for cell replacement therapy. In particular, the effect of mechanical stress on differentiation is becoming attractive for the field of regenerative medicine. Therefore, the development of a stress-mediated differentiation protocol is an important matter for the future.
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Souidi M, Sleiman Y, Acimovic I, Pribyl J, Charrabi A, Baecker V, Scheuermann V, Pesl M, Jelinkova S, Skladal P, Dvorak P, Lacampagne A, Rotrekl V, Meli AC. Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids. Int J Mol Sci 2021; 22:ijms22020662. [PMID: 33440843 PMCID: PMC7827232 DOI: 10.3390/ijms22020662] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 12/22/2020] [Accepted: 01/06/2021] [Indexed: 02/07/2023] Open
Abstract
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
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Affiliation(s)
- Monia Souidi
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
| | - Yvonne Sleiman
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
| | - Ivana Acimovic
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
- Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; (S.J.); (P.D.); (V.R.)
| | - Jan Pribyl
- CEITEC, Masaryk University, 62500 Brno, Czech Republic; (J.P.); (P.S.)
| | - Azzouz Charrabi
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
| | - Volker Baecker
- Montpellier Ressources Imagerie, BioCampus Montpellier, CNRS, INSERM, University of Montpellier, 34000 Montpellier, France;
| | - Valerie Scheuermann
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
| | - Martin Pesl
- Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; (S.J.); (P.D.); (V.R.)
- International Clinical Research Center, St. Anne’s University Hospital Brno, 65691 Brno, Czech Republic
- First Department of Internal Medicine/Cardioangiology, St. Anne’s Hospital, Masaryk University, 65691 Brno, Czech Republic
- Correspondence: (M.P.); (A.C.M.); Tel.: +420-723-860-905 (M.P.); +33-4-67-41-52-44 (A.C.M.); Fax: +33-4-67-41-52-42 (A.C.M.)
| | - Sarka Jelinkova
- Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; (S.J.); (P.D.); (V.R.)
| | - Petr Skladal
- CEITEC, Masaryk University, 62500 Brno, Czech Republic; (J.P.); (P.S.)
| | - Petr Dvorak
- Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; (S.J.); (P.D.); (V.R.)
| | - Alain Lacampagne
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
| | - Vladimir Rotrekl
- Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; (S.J.); (P.D.); (V.R.)
- International Clinical Research Center, St. Anne’s University Hospital Brno, 65691 Brno, Czech Republic
| | - Albano C. Meli
- PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France; (M.S.); (Y.S.); (I.A.); (A.C.); (V.S.); (A.L.)
- Correspondence: (M.P.); (A.C.M.); Tel.: +420-723-860-905 (M.P.); +33-4-67-41-52-44 (A.C.M.); Fax: +33-4-67-41-52-42 (A.C.M.)
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Farzaneh Z, Abbasalizadeh S, Asghari-Vostikolaee MH, Alikhani M, Cabral JMS, Baharvand H. Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor. Biotechnol Bioeng 2020; 117:3739-3756. [PMID: 32725885 DOI: 10.1002/bit.27521] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2020] [Revised: 07/11/2020] [Accepted: 07/27/2020] [Indexed: 12/11/2022]
Abstract
Developing technologies for scalable production of human organoids has gained increased attention for "organoid medicine" and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20-40% dissolved oxygen concentration [DO] (corresponded to 30-60 mmHg pO2 within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.
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Affiliation(s)
- Zahra Farzaneh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Institute Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Mohammad-Hassan Asghari-Vostikolaee
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mehdi Alikhani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Joaquim M S Cabral
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Institute Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
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Ashok P, Parikh A, Du C, Tzanakakis ES. Xenogeneic-Free System for Biomanufacturing of Cardiomyocyte Progeny From Human Pluripotent Stem Cells. Front Bioeng Biotechnol 2020; 8:571425. [PMID: 33195131 PMCID: PMC7644809 DOI: 10.3389/fbioe.2020.571425] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 09/28/2020] [Indexed: 01/14/2023] Open
Abstract
Functional heart cells and tissues sourced from human pluripotent stem cells (hPSCs) have great potential for substantially advancing treatments of cardiovascular maladies. Realization of this potential will require the development of cost-effective and tunable bioprocesses for manufacturing hPSC-based cell therapeutics. Here, we report the development of a xeno-free platform for guiding the cardiogenic commitment of hPSCs. The system is based on a fully defined, open-source formulation without complex supplements, which have varied and often undetermined effects on stem cell physiology. The formulation was used to systematically investigate factors inducing the efficient commitment to cardiac mesoderm of three hPSC lines. Contractile clusters of cells appeared within a week of differentiation in planar cultures and by day 13 over 80% of the cells expressed cardiac progeny markers such as TNNT2. In conjunction with expansion, this differentiation strategy was employed in stirred-suspension cultures of hPSCs. Scalable differentiation resulted in 0.4-2 million CMs/ml or ∼5-20 TNNT2-positive cells per seeded hPSC without further enrichment. Our findings will contribute to the engineering of bioprocesses advancing the manufacturing of stem cell-based therapeutics for heart diseases.
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Affiliation(s)
- Preeti Ashok
- Chemical and Biological Engineering, Tufts University, Medford, MA, United States
| | | | - Chuang Du
- Biomedical Engineering, Tufts University, Medford, MA, United States
| | - Emmanuel S. Tzanakakis
- Chemical and Biological Engineering, Tufts University, Medford, MA, United States
- Clinical and Translational Science Institute, Tufts Medical Center, Boston, MA, United States
- Developmental Molecular and Chemical Biology, Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, United States
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Patino-Guerrero A, Veldhuizen J, Zhu W, Migrino RQ, Nikkhah M. Three-dimensional scaffold-free microtissues engineered for cardiac repair. J Mater Chem B 2020; 8:7571-7590. [PMID: 32724973 PMCID: PMC8314954 DOI: 10.1039/d0tb01528h] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Cardiovascular diseases, including myocardial infarction (MI), persist as the leading cause of mortality and morbidity worldwide. The limited regenerative capacity of the myocardium presents significant challenges specifically for the treatment of MI and, subsequently, heart failure (HF). Traditional therapeutic approaches mainly rely on limiting the induced damage or the stress on the remaining viable myocardium through pharmacological regulation of remodeling mechanisms, rather than replacement or regeneration of the injured tissue. The emerging alternative regenerative medicine-based approaches have focused on restoring the damaged myocardial tissue with newly engineered functional and bioinspired tissue units. Cardiac regenerative medicine approaches can be broadly categorized into three groups: cell-based therapies, scaffold-based cardiac tissue engineering, and scaffold-free cardiac tissue engineering. Despite significant advancements, however, the clinical translation of these approaches has been critically hindered by two key obstacles for successful structural and functional replacement of the damaged myocardium, namely: poor engraftment of engineered tissue into the damaged cardiac muscle and weak electromechanical coupling of transplanted cells with the native tissue. To that end, the integration of micro- and nanoscale technologies along with recent advancements in stem cell technologies have opened new avenues for engineering of structurally mature and highly functional scaffold-based (SB-CMTs) and scaffold-free cardiac microtissues (SF-CMTs) with enhanced cellular organization and electromechanical coupling for the treatment of MI and HF. In this review article, we will present the state-of-the-art approaches and recent advancements in the engineering of SF-CMTs for myocardial repair.
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18
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Branco MA, Cabral JM, Diogo MM. From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges. Bioengineering (Basel) 2020; 7:E92. [PMID: 32785039 PMCID: PMC7552661 DOI: 10.3390/bioengineering7030092] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/30/2020] [Accepted: 08/06/2020] [Indexed: 12/19/2022] Open
Abstract
The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.
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Affiliation(s)
| | | | - Maria Margarida Diogo
- iBB-Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; (M.A.B.); (J.M.S.C.)
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19
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Laco F, Lam ATL, Woo TL, Tong G, Ho V, Soong PL, Grishina E, Lin KH, Reuveny S, Oh SKW. Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor. Stem Cell Res Ther 2020; 11:118. [PMID: 32183888 PMCID: PMC7076930 DOI: 10.1186/s13287-020-01618-6] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 02/11/2020] [Accepted: 02/24/2020] [Indexed: 01/13/2023] Open
Abstract
Background The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Methods Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Results Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. Conclusion A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry. Supplementary information The online version of this article (10.1186/s13287-020-01618-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Filip Laco
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Alan Tin-Lun Lam
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
| | - Tsung-Liang Woo
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Gerine Tong
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Valerie Ho
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Poh-Loong Soong
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Elina Grishina
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Kun-Han Lin
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Shaul Reuveny
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Steve Kah-Weng Oh
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
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Jiang B, Yan L, Shamul JG, Hakun M, He X. Stem cell therapy of myocardial infarction: a promising opportunity in bioengineering. ADVANCED THERAPEUTICS 2020; 3:1900182. [PMID: 33665356 PMCID: PMC7928435 DOI: 10.1002/adtp.201900182] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Indexed: 02/06/2023]
Abstract
Myocardial infarction (MI) is a life-threatening disease resulting from irreversible death of cardiomyocytes (CMs) and weakening of the heart blood-pumping function. Stem cell-based therapies have been studied for MI treatment over the last two decades with promising outcome. In this review, we critically summarize the past work in this field to elucidate the advantages and disadvantages of treating MI using pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), adult stem cells, and cardiac progenitor cells. The main advantage of the latter is their cytokine production capability to modulate immune responses and control the progression of healing. However, human adult stem cells have very limited (if not 'no') capacity to differentiate into functional CMs in vitro or in vivo. In contrast, PSCs can be differentiated into functional CMs although the protocols for the cardiac differentiation of PSCs are mainly for adherent cells under 2D culture. Derivation of PSC-CMs in 3D, allowing for large-scale production of CMs via modulation of the Wnt/β-catenin signal pathway with defined chemicals and medium, may be desired for clinical translation. Furthermore, the technology of purification and maturation of the PSC-CMs may need further improvements to eliminate teratoma formation after in vivo implantation of the PSC-CMs for treating MI. In addition, in vitro derived PSC-CMs may have mechanical and electrical mismatch with the patient's cardiac tissue, which causes arrhythmia. This supports the use of PSC-derived cells committed to cardiac lineage without beating for implantation to treat MI. In this case, the PSC derived cells may utilize the mechanical, electrical, and chemical cues in the heart to further differentiate into mature/functional CMs in situ. Another major challenge facing stem cell therapy of MI is the low retention/survival of stem cells or their derivatives (e.g., PSC-CMs) in the heart for MI treatment after injection in vivo. This may be resolved by using biomaterials to engineer stem cells for reduced immunogenicity, immobilization of the cells in the heart, and increased integration with the host cardiac tissue. Biomaterials have also been applied in the derivation of CMs in vitro to increase the efficiency and maturation of differentiation. Collectively, a lot has been learned from the past failure of simply injecting intact stem cells or their derivatives in vivo for treating MI, and bioengineering stem cells with biomaterials is expected to be a valuable strategy for advancing stem cell therapy towards its widespread application for treating MI in the clinic.
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Affiliation(s)
- Bin Jiang
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Li Yan
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - James G Shamul
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Maxwell Hakun
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Xiaoming He
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
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21
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An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations. Sci Rep 2019; 9:15655. [PMID: 31666563 PMCID: PMC6821886 DOI: 10.1038/s41598-019-51944-w] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2018] [Accepted: 10/08/2019] [Indexed: 01/14/2023] Open
Abstract
Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1–3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.
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Branco MA, Cotovio JP, Rodrigues CAV, Vaz SH, Fernandes TG, Moreira LM, Cabral JMS, Diogo MM. Transcriptomic analysis of 3D Cardiac Differentiation of Human Induced Pluripotent Stem Cells Reveals Faster Cardiomyocyte Maturation Compared to 2D Culture. Sci Rep 2019; 9:9229. [PMID: 31239450 PMCID: PMC6592905 DOI: 10.1038/s41598-019-45047-9] [Citation(s) in RCA: 76] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2018] [Accepted: 05/30/2019] [Indexed: 12/11/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT+ cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.
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Affiliation(s)
- Mariana A Branco
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - João P Cotovio
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - Carlos A V Rodrigues
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - Sandra H Vaz
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028, Lisbon, Portugal.,Instituto de Farmacologia e Neurociências, Faculdade de Medicina da Universidade de Lisboa, 1649-028, Lisbon, Portugal
| | - Tiago G Fernandes
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - Leonilde M Moreira
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
| | - Maria Margarida Diogo
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal. .,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
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Cha JM, Lee MY, Hong J. Bioreactor systems are essentially required for stem cell bioprocessing. PRECISION AND FUTURE MEDICINE 2019. [DOI: 10.23838/pfm.2018.00128] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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24
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Human Pluripotent Stem Cells: Applications and Challenges for Regenerative Medicine and Disease Modeling. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2019; 171:189-224. [PMID: 31740987 DOI: 10.1007/10_2019_117] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In recent years, human pluripotent stem (hPS) cells have started to emerge as a potential tool with application in fields such as regenerative medicine, disease modeling, and drug screening. In particular, the ability to differentiate human-induced pluripotent stem (hiPS) cells into different cell types and to mimic structures and functions of a specific target organ, resourcing to organoid technology, has introduced novel model systems for disease recapitulation while offering a powerful tool to provide a faster and reproducible approach in the process of drug discovery. All these technologies are expected to improve the overall quality of life of the humankind. Here, we highlight the main applications of hiPS cells and the main challenges associated with the translation of hPS cell derivatives into clinical settings and other biomedical applications, such as the costs of the process and the ability to mimic the complexity of the in vivo systems. Moreover, we focus on the bioprocessing approaches that can be applied towards the production of high numbers of cells as well as their efficient differentiation into the final product and further purification.
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25
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Fernández-Morales JC, Hua W, Yao Y, Morad M. Regulation of Ca 2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells. Cell Calcium 2018; 78:1-14. [PMID: 30579812 DOI: 10.1016/j.ceca.2018.12.006] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2018] [Revised: 12/11/2018] [Accepted: 12/11/2018] [Indexed: 12/20/2022]
Abstract
AIMS The effects of acute (100 s) hypoxia and/or acidosis on Ca2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time. METHODS AND RESULTS 1) hiPSC-CMs express two cell populations: rapidly-inactivating ICa myocytes (τi<40 ms, in 4-5 day cultures) and slowly-inactivating ICa (τi ≥ 40 ms, in 6-8 day cultures). 2) Hypoxia suppressed ICa by 10-20% in rapidly- and 40-55% in slowly-inactivating ICa cells. 3) Isoproterenol enhanced ICa in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba2+ was the charge carrier through the calcium channels, implicating Ca2+-dependent inactivation in O2 sensing. 5) Acidosis suppressed ICa by ∼35% and ∼25% in rapidly and slowly inactivating ICa cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca2+-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. CONCLUSIONS Variability in ICa inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca2+-signaling in the two-cell types arises from differential Ca2+-dependent inactivation of the Ca2+-channel caused by proximity of Ca2+-release stores to the Ca2+ channels.
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Affiliation(s)
| | - Wei Hua
- Cardiac Signaling Center of MUSC, USC and Clemson, Charleston, SC, USA
| | - Yuyu Yao
- Cardiac Signaling Center of MUSC, USC and Clemson, Charleston, SC, USA
| | - Martin Morad
- Cardiac Signaling Center of MUSC, USC and Clemson, Charleston, SC, USA; Department of Pharmacology,Georgetown University Medical Center, Washington, DC, USA.
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26
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Ting S, Lam A, Tong G, Chen A, Wei H, Wu J, Lam YN, Reuveny S, Oh S. Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess. Stem Cell Res 2018; 31:161-173. [DOI: 10.1016/j.scr.2018.07.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Revised: 07/12/2018] [Accepted: 07/23/2018] [Indexed: 01/15/2023] Open
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27
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Abou-Saleh H, Zouein FA, El-Yazbi A, Sanoudou D, Raynaud C, Rao C, Pintus G, Dehaini H, Eid AH. The march of pluripotent stem cells in cardiovascular regenerative medicine. Stem Cell Res Ther 2018; 9:201. [PMID: 30053890 PMCID: PMC6062943 DOI: 10.1186/s13287-018-0947-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.
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Affiliation(s)
- Haissam Abou-Saleh
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
| | - Fouad A. Zouein
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ahmed El-Yazbi
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Pharmacology and Toxicology, Alexandria University, Alexandria, Egypt
| | - Despina Sanoudou
- Clinical Genomics and Pharmacogenomics Unit, 4th Department of Internal Medicine, “Attikon” Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | | | - Christopher Rao
- Department of Surgery, Queen Elizabeth Hospital, Woolwich, London, UK
| | - Gianfranco Pintus
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
| | - Hassan Dehaini
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ali H. Eid
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
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28
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Ovando-Roche P, West EL, Branch MJ, Sampson RD, Fernando M, Munro P, Georgiadis A, Rizzi M, Kloc M, Naeem A, Ribeiro J, Smith AJ, Gonzalez-Cordero A, Ali RR. Use of bioreactors for culturing human retinal organoids improves photoreceptor yields. Stem Cell Res Ther 2018; 9:156. [PMID: 29895313 PMCID: PMC5998504 DOI: 10.1186/s13287-018-0907-0] [Citation(s) in RCA: 81] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Revised: 04/30/2018] [Accepted: 05/15/2018] [Indexed: 11/28/2022] Open
Abstract
Background The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. Methods We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. Results Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. Conclusions Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies. Electronic supplementary material The online version of this article (10.1186/s13287-018-0907-0) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Patrick Ovando-Roche
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Emma L West
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Matthew J Branch
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Robert D Sampson
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Milan Fernando
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Peter Munro
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Anastasios Georgiadis
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Matteo Rizzi
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Magdalena Kloc
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Arifa Naeem
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Joana Ribeiro
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Alexander J Smith
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Anai Gonzalez-Cordero
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK
| | - Robin R Ali
- Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK. .,NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London, EC1V 2PD, UK.
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Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications. Cells 2018; 7:cells7060048. [PMID: 29799480 PMCID: PMC6025241 DOI: 10.3390/cells7060048] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Revised: 05/16/2018] [Accepted: 05/22/2018] [Indexed: 12/11/2022] Open
Abstract
Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications.
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Rattananinsruang P, Dechsukhum C, Leeanansaksiri W. Establishment of Insulin-Producing Cells From Human Embryonic Stem Cells Underhypoxic Condition for Cell Based Therapy. Front Cell Dev Biol 2018; 6:49. [PMID: 29868580 PMCID: PMC5962719 DOI: 10.3389/fcell.2018.00049] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 04/16/2018] [Indexed: 12/27/2022] Open
Abstract
Diabetes mellitus (DM) is a group of diseases characterized by abnormally high levels of glucose in the blood stream. In developing a potential therapy for diabetic patients, pancreatic cells transplantation has drawn great attention. However, the hinder of cell transplantation for diabetes treatment is insufficient sources of insulin-producing cells. Therefore, new cell based therapy need to be developed. In this regard, human embryonic stem cells (hESCs) may serve as good candidates for this based on their capability of differentiation into various cell types. In this study, we designed a new differentiation protocol that can generate hESC-derived insulin-producing cells (hES-DIPCs) in a hypoxic condition. We also emphasized on the induction of definitive endoderm during embryoid bodies (EBs) formation. After induction of hESCs differentiation into insulin-producing cells (IPCs), the cells obtained from the cultures exhibited pancreas-related genes such as Pdx1, Ngn3, Nkx6.1, GLUT2, and insulin. These cells also showed positive for DTZ-stained cellular clusters and contained ability of insulin secretion in a glucose-dependent manner. After achievement to generated functional hES-DIPCs in vitro, some of the hES-DIPCs were then encapsulated named encapsulated hES-DIPCs. The data showed that the encapsulated cells could possess the function of insulin secretion in a time-dependent manner. The hES-DIPCs and encapsulated hES-DIPCs were then separately transplanted into STZ-induced diabetic mice. The findings showed the significant blood glucose levels regulation capacity and declination of IL-1β concentration in all transplanted mice. These results indicated that both hES-DIPCs and encapsulated hES-DIPCs contained the ability to sustain hyperglycemia condition as well as decrease inflammatory cytokine level in vivo. The findings of this study may apply for generation of a large number of hES-DIPCs in vitro. In addition, the implication of this work is therapeutic value in type I diabetes treatment in the future. The application for type II diabetes treatment remain to be investigated.
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Affiliation(s)
- Piyaporn Rattananinsruang
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Chavaboon Dechsukhum
- School of Pathology, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Wilairat Leeanansaksiri
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
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Ke Y, Liu C, Wang Y, Xiao M, Fan J, Fu P, Wang S, Wu G. Cell-loaded carboxymethylcellulose microspheres sustain viability and proliferation of ATDC5 cells. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2018; 46:140-151. [DOI: 10.1080/21691401.2018.1452751] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Affiliation(s)
- Yu Ke
- Department of Biomedical Engineering, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, College of Life Science and Technology, Jinan University, Guangzhou, China
| | - Caikun Liu
- Department of Biomedical Engineering, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, College of Life Science and Technology, Jinan University, Guangzhou, China
| | - Yanting Wang
- Department of Biomedical Engineering, South China University of Technology, Guangzhou, China
| | - Meng Xiao
- Department of Materials Science and Engineering, School of Chemistry and Materials, Jinan University, Guangzhou, China
| | - Jiachen Fan
- Department of Biomedical Engineering, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, College of Life Science and Technology, Jinan University, Guangzhou, China
| | - Pengcheng Fu
- Department of Biomedical Engineering, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, College of Life Science and Technology, Jinan University, Guangzhou, China
| | - Shuhao Wang
- Department of Biomedical Engineering, South China University of Technology, Guangzhou, China
| | - Gang Wu
- Department of Biomedical Engineering, South China University of Technology, Guangzhou, China
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Tohyama S, Fujita J, Fujita C, Yamaguchi M, Kanaami S, Ohno R, Sakamoto K, Kodama M, Kurokawa J, Kanazawa H, Seki T, Kishino Y, Okada M, Nakajima K, Tanosaki S, Someya S, Hirano A, Kawaguchi S, Kobayashi E, Fukuda K. Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes. Stem Cell Reports 2017; 9:1406-1414. [PMID: 28988990 PMCID: PMC5829307 DOI: 10.1016/j.stemcr.2017.08.025] [Citation(s) in RCA: 87] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Revised: 08/30/2017] [Accepted: 08/31/2017] [Indexed: 01/08/2023] Open
Abstract
Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.
Efficient mass production of hiPSCs by multilayer culture plates with AGV Efficient mass production of hiPSC-CMs using a massive 2D culture system with AGV Mass production of pure hiPSC-CMs via metabolic selection
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Affiliation(s)
- Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
| | - Chihana Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Miho Yamaguchi
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sayaka Kanaami
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Rei Ohno
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kazuho Sakamoto
- Department of Pharmacology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan; Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Masami Kodama
- Department of Bio-informational Pharmacology, Medical Research Institute, National University Corporation Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Junko Kurokawa
- Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Hideaki Kanazawa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Tomohisa Seki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kazuaki Nakajima
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sho Tanosaki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shota Someya
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Akinori Hirano
- Department of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shinji Kawaguchi
- Department of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Eiji Kobayashi
- Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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Sart S, Bejoy J, Li Y. Characterization of 3D pluripotent stem cell aggregates and the impact of their properties on bioprocessing. Process Biochem 2017. [DOI: 10.1016/j.procbio.2016.05.024] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Xu X, Wang W, Li Z, Kratz K, Ma N, Lendlein A. Surface geometry of poly(ether imide) boosts mouse pluripotent stem cell spontaneous cardiomyogenesis via modulating the embryoid body formation process. Clin Hemorheol Microcirc 2017; 64:367-382. [DOI: 10.3233/ch-168107] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Affiliation(s)
- Xun Xu
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Weiwei Wang
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
| | - Zhengdong Li
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Karl Kratz
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
| | - Nan Ma
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
| | - Andreas Lendlein
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
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35
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Galvanauskas V, Grincas V, Simutis R, Kagawa Y, Kino-oka M. Current state and perspectives in modeling and control of human pluripotent stem cell expansion processes in stirred-tank bioreactors. Biotechnol Prog 2017; 33:355-364. [DOI: 10.1002/btpr.2431] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2016] [Revised: 12/10/2016] [Indexed: 01/02/2023]
Affiliation(s)
| | - Vykantas Grincas
- Department of Automation; Kaunas University of Technology; Kaunas Lithuania
| | - Rimvydas Simutis
- Department of Automation; Kaunas University of Technology; Kaunas Lithuania
| | - Yuki Kagawa
- Department of Biotechnology; Osaka University; Osaka Japan
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36
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Kang J, Kim TW, Hur J, Kim HS. Strategy to Prime the Host and Cells to Augment Therapeutic Efficacy of Progenitor Cells for Patients with Myocardial Infarction. Front Cardiovasc Med 2016; 3:46. [PMID: 27933299 PMCID: PMC5121226 DOI: 10.3389/fcvm.2016.00046] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Accepted: 11/08/2016] [Indexed: 11/23/2022] Open
Abstract
Cell therapy in myocardial infarction (MI) is an innovative strategy that is regarded as a rescue therapy to repair the damaged myocardium and to promote neovascularization for the ischemic border zone. Among several stem cell sources for this purpose, autologous progenitors from bone marrow or peripheral blood would be the most feasible and safest cell-source. Despite the theoretical benefit of cell therapy, this method is not widely adopted in the actual clinical practice due to its low therapeutic efficacy. Various methods have been used to augment the efficacy of cell therapy in MI, such as using different source of progenitors, genetic manipulation of cells, or priming of the cells or hosts (patients) with agents. Among these methods, the strategy to augment the therapeutic efficacy of the autologous peripheral blood mononuclear cells (PBMCs) by priming agents may be the most feasible and the safest method that can be applied directly to the clinic. In this review, we will discuss the current status and future directions of priming PBMCs or patients, as for cell therapy of MI.
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Affiliation(s)
- Jeehoon Kang
- Department of Medicine, Seoul National University Hospital, Seoul, South Korea; Molecular Medicine & Biopharmaceutical Science, Graduate School of Convergence Science & Technology, Seoul National University, Seoul, South Korea
| | - Tae-Won Kim
- Molecular Medicine & Biopharmaceutical Science, Graduate School of Convergence Science & Technology, Seoul National University, Seoul, South Korea; National Research Laboratory for Stem Cell Niche, Center for Medical Innovation, Seoul National University Hospital, Seoul, South Korea
| | - Jin Hur
- National Research Laboratory for Stem Cell Niche, Center for Medical Innovation, Seoul National University Hospital , Seoul , South Korea
| | - Hyo-Soo Kim
- Department of Medicine, Seoul National University Hospital, Seoul, South Korea; Molecular Medicine & Biopharmaceutical Science, Graduate School of Convergence Science & Technology, Seoul National University, Seoul, South Korea; National Research Laboratory for Stem Cell Niche, Center for Medical Innovation, Seoul National University Hospital, Seoul, South Korea
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37
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Sharma VS, Khalife R, Tostoes R, Leung L, Kinsella R, Ruban L, Veraitch FS. Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures. Biotechnol Lett 2016; 39:339-350. [PMID: 27812821 PMCID: PMC5247545 DOI: 10.1007/s10529-016-2244-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Accepted: 10/27/2016] [Indexed: 11/17/2022]
Abstract
Objective To develop a microwell suspension platform for the adaption of attached stem cell differentiation protocols into mixed suspension culture. Results We adapted an adherent protocol for the retinal differentiation of human induced pluripotent stem cells (hiPSCs) using a two-step protocol. Establishing the optimum embryoid body (EB) starting size and shaking speed resulted in the translation of the original adherent process into suspension culture. Embryoid bodies expanded in size as the culture progressed resulting in the expression of characteristic markers of early (Rx, Six and Otx2) and late (Crx, Nrl and Rhodopsin) retinal differentiation. The new process also eliminated the use of matrigel, an animal-derived extracellular matrix coating. Conclusions Shaking microwells offer a fast and cost-effective method for proof-of-concept studies to establish whether pluripotent stem cell differentiation processes can be translated into mixed suspension culture. Electronic supplementary material The online version of this article (doi:10.1007/s10529-016-2244-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Vishal S Sharma
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Rana Khalife
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Rui Tostoes
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Leonard Leung
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Rose Kinsella
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Ludmilla Ruban
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK
| | - Farlan S Veraitch
- Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
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38
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Yang D, Wang J, Xiao M, Zhou T, Shi X. Role of Mir-155 in Controlling HIF-1α Level and Promoting Endothelial Cell Maturation. Sci Rep 2016; 6:35316. [PMID: 27731397 PMCID: PMC5059686 DOI: 10.1038/srep35316] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2016] [Accepted: 09/28/2016] [Indexed: 01/03/2023] Open
Abstract
Stem-cell-based therapy for cardiovascular disease, especially ischemic heart disease (IHD), is a promising approach to facilitating neovascularization through the migration of stem cells to the ischemic site and their subsequent differentiation into endothelial cells (ECs). Hypoxia is a chief feature of IHD and the stem cell niche. However, whether hypoxia promotes stem cell differentiation into ECs or causes them to retain their stemness is controversial. Here, the differentiation of pluripotent stem cells (iPSCs) into endothelial cells (ECs) was induced under hypoxia. Though the angiogenic capability and angiogenesis-related autocrine/paracrine factors of the ECs were improved under hypoxia, the level of hypoxia inducible factor 1α (HIF-1α) was nonetheless found to be restricted along with the EC differentiation. The down-regulation of HIF-1α was found to have been caused by VEGF-induced microRNA-155 (miR-155). Moreover, miR-155 was also found to enhance the angiogenic capability of induced ECs by targeting E2F2 transcription factor. Hence, miR-155 not only contributes to controlling HIF-1α expression under hypoxia but also promotes angiogenesis, which is a key feature of mature ECs. Revealing the real role of hypoxia and clarifying the function of miR-155 in EC differentiation may facilitate improvement of angiogenic gene- and stem-cell-based therapies for ischemic heart disease.
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Affiliation(s)
- Deguang Yang
- Department of Cardiology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510000, China
| | - Jinhong Wang
- Department of Respiration, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510000, China
| | - Meng Xiao
- Department of Nursing, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510000, China
| | - Tao Zhou
- Department of Cardiology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510000, China
| | - Xu Shi
- Central Laboratory, the First Hospital of Jilin University, Changchun, 130032, China
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39
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Bang OY, Kim EH, Cha JM, Moon GJ. Adult Stem Cell Therapy for Stroke: Challenges and Progress. J Stroke 2016; 18:256-266. [PMID: 27733032 PMCID: PMC5066440 DOI: 10.5853/jos.2016.01263] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2016] [Revised: 09/15/2016] [Accepted: 09/18/2016] [Indexed: 02/06/2023] Open
Abstract
Stroke is one of the leading causes of death and physical disability among adults. It has been 15 years since clinical trials of stem cell therapy in patients with stroke have been conducted using adult stem cells like mesenchymal stem cells and bone marrow mononuclear cells. Results of randomized controlled trials showed that adult stem cell therapy was safe but its efficacy was modest, underscoring the need for new stem cell therapy strategies. The primary limitations of current stem cell therapies include (a) the limited source of engraftable stem cells, (b) the presence of optimal time window for stem cell therapies, (c) inherited limitation of stem cells in terms of growth, trophic support, and differentiation potential, and (d) possible transplanted cell-mediated adverse effects, such as tumor formation. Here, we discuss recent advances that overcome these hurdles in adult stem cell therapy for stroke.
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Affiliation(s)
- Oh Young Bang
- Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.,Translational and Stem Cell Research Laboratory on Stroke, Samsung Medical Center, Seoul, Korea
| | - Eun Hee Kim
- Translational and Stem Cell Research Laboratory on Stroke, Samsung Medical Center, Seoul, Korea
| | - Jae Min Cha
- Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Co., Ltd., Seoul, Korea.,Medical Device Research Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Korea
| | - Gyeong Joon Moon
- Translational and Stem Cell Research Laboratory on Stroke, Samsung Medical Center, Seoul, Korea.,Stem cell and Regenerative Medicine Institute, Samsung Biomedical Research Institute, Seoul, Korea
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40
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Bauwens CL, Toms D, Ungrin M. Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells. J Vis Exp 2016. [PMID: 27768032 PMCID: PMC5092056 DOI: 10.3791/54308] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.
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Affiliation(s)
- Celine L Bauwens
- Institute of Biomaterials and Biomedical Engineering, University of Toronto
| | - Derek Toms
- Department of Comparative Biology and Experimental Medicine, University of Calgary
| | - Mark Ungrin
- Department of Comparative Biology and Experimental Medicine, University of Calgary;
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41
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Wei R, Yang J, Gao M, Wang H, Hou W, Mu Y, Chen G, Hong T. Infarcted cardiac microenvironment may hinder cardiac lineage differentiation of human embryonic stem cells. Cell Biol Int 2016; 40:1235-1246. [PMID: 27600481 DOI: 10.1002/cbin.10679] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2016] [Accepted: 09/04/2016] [Indexed: 11/07/2022]
Abstract
Microenvironment regulates cell fate and function. In this study, we investigated the effects of the infarcted cardiac microenvironment on cardiac differentiation of human embryonic stem cells (hESCs). hESCs were intramyocardially transplanted into infarcted or uninjured rat hearts. After 4 weeks, mesodermal and cardiac lineage markers were detected by immunofluorescence. Cardiac function was assessed by echocardiography. hESCs were differentiated in vitro under hypoxic (5% O2 ), low-nutrient (5% FBS), or control condition. The numbers of beating clusters, proportions of cardiac troponin T (cTnT)-positive cells, and relative levels of cardiac-specific markers were determined. Results showed that in both uninjured and infarcted hearts, hESCs survived, underwent development, and formed intracardiac grafts, with a higher proportion in the uninjured hearts. However, cells that were double positive for human fetal liver kinase 1 (Flk1), a marker of cardiac progenitors, and human β-tubulin, a marker for labeling human cells, were found in the uninjured hearts but not in the infarcted hearts. hESC transplantation did not restore the cardiac function of acutely infarcted rats. In vitro, low FBS treatment was associated with fewer beating clusters, a lower proportion of cTnT-positive cells and lower levels of cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) expression than those in the control. Conversely, hypoxia treatment was associated with a higher proportion of cTnT-positive cells and higher levels of cTnI expression. In conclusion, transplanted hESCs differentiate toward Flk1-positive cardiac progenitors in the uninjured but not infarcted hearts. The infarcted cardiac microenvironment recapitulated is unsuitable for cardiac differentiation of hESCs, likely due to nutrient deprivation.
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Affiliation(s)
- Rui Wei
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China.,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Jin Yang
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China.,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Meijuan Gao
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China.,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Haining Wang
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China.,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Wenfang Hou
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China.,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Yiming Mu
- Department of Endocrinology, Chinese PLA General Hospital, Beijing, China
| | - Guian Chen
- Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China
| | - Tianpei Hong
- Department of Endocrinology and Metabolism, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191, China. .,Clinical Stem Cell Research Centre, Peking University Third Hospital, Beijing, China.
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42
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Fonoudi H, Ansari H, Abbasalizadeh S, Blue GM, Aghdami N, Winlaw DS, Harvey RP, Bosman A, Baharvand H. Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol. J Vis Exp 2016. [PMID: 27500408 DOI: 10.3791/54276] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.
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Affiliation(s)
- Hananeh Fonoudi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Hassan Ansari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR
| | - Gillian M Blue
- Heart Centre for Children, The Children´s Hospital at Westmead; Sydney Medical School, University of Sydney
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR
| | - David S Winlaw
- Heart Centre for Children, The Children´s Hospital at Westmead; Sydney Medical School, University of Sydney
| | - Richard P Harvey
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales; School of Biotechnology and Biomolecular Sciences, University of New South Wales
| | - Alexis Bosman
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales;
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Department of Developmental Biology, University of Science and Culture
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43
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White DE, Sylvester JB, Levario TJ, Lu H, Streelman JT, McDevitt TC, Kemp ML. Quantitative multivariate analysis of dynamic multicellular morphogenic trajectories. Integr Biol (Camb) 2016; 7:825-33. [PMID: 26095427 DOI: 10.1039/c5ib00072f] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.
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Affiliation(s)
- Douglas E White
- The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
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44
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Lewandowski J, Kolanowski TJ, Kurpisz M. Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes. J Tissue Eng Regen Med 2016; 11:1658-1674. [PMID: 26777594 DOI: 10.1002/term.2117] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Revised: 09/16/2015] [Accepted: 11/18/2015] [Indexed: 01/04/2023]
Abstract
The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post-infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC-mediated cardiomyocytes (iPSC-CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless-type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co-cultures with visceral endoderm-like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well-suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm-like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non-genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Jarosław Lewandowski
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Tomasz J Kolanowski
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Maciej Kurpisz
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
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45
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Kempf H, Andree B, Zweigerdt R. Large-scale production of human pluripotent stem cell derived cardiomyocytes. Adv Drug Deliv Rev 2016; 96:18-30. [PMID: 26658242 DOI: 10.1016/j.addr.2015.11.016] [Citation(s) in RCA: 78] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Revised: 11/19/2015] [Accepted: 11/25/2015] [Indexed: 12/20/2022]
Abstract
Regenerative medicine, including preclinical studies in large animal models and tissue engineering approaches as well as innovative assays for drug discovery, will require the constant supply of hPSC-derived cardiomyocytes and other functional progenies. Respective cell production processes must be robust, economically viable and ultimately GMP-compliant. Recent research has enabled transition of lab scale protocols for hPSC expansion and cardiomyogenic differentiation towards more controlled processing in industry-compatible culture platforms. Here, advanced strategies for the cultivation and differentiation of hPSCs will be reviewed by focusing on stirred bioreactor-based techniques for process upscaling. We will discuss how cardiomyocyte mass production might benefit from recent findings such as cell expansion at the cardiovascular progenitor state. Finally, remaining challenges will be highlighted, specifically regarding three dimensional (3D) hPSC suspension culture and critical safety issues ahead of clinical translation.
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Affiliation(s)
- Henning Kempf
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, Hannover Medical School, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Birgit Andree
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, Hannover Medical School, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Robert Zweigerdt
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, Hannover Medical School, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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46
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Hammoud AA, Kirstein N, Mournetas V, Darracq A, Broc S, Blanchard C, Zeineddine D, Mortada M, Boeuf H. Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia. PLoS One 2016; 11:e0146281. [PMID: 26731538 PMCID: PMC4701481 DOI: 10.1371/journal.pone.0146281] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 12/15/2015] [Indexed: 12/12/2022] Open
Abstract
Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF-mediated reprogramming are poorly understood. By employing a LIF-dependent 'plasticity' test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of Pluri and Master gene expression in comparison to 20% O2.
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Affiliation(s)
- Aya Abou Hammoud
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
- Lebanese University, Beyrouth, Liban
| | - Nina Kirstein
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Virginie Mournetas
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Anais Darracq
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Sabine Broc
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Camille Blanchard
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | | | | | - Helene Boeuf
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
- * E-mail:
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Validation of Bioreactor and Human-on-a-Chip Devices for Chemical Safety Assessment. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 856:299-316. [PMID: 27671728 DOI: 10.1007/978-3-319-33826-2_12] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Equipment and device qualification and test assay validation in the field of tissue engineered human organs for substance assessment remain formidable tasks with only a few successful examples so far. The hurdles seem to increase with the growing complexity of the biological systems, emulated by the respective models. Controlled single tissue or organ culture in bioreactors improves the organ-specific functions and maintains their phenotypic stability for longer periods of time. The reproducibility attained with bioreactor operations is, per se, an advantage for the validation of safety assessment. Regulatory agencies have gradually altered the validation concept from exhaustive "product" to rigorous and detailed process characterization, valuing reproducibility as a standard for validation. "Human-on-a-chip" technologies applying micro-physiological systems to the in vitro combination of miniaturized human organ equivalents into functional human micro-organisms are nowadays thought to be the most elaborate solution created to date. They target the replacement of the current most complex models-laboratory animals. Therefore, we provide here a road map towards the validation of such "human-on-a-chip" models and qualification of their respective bioreactor and microchip equipment along a path currently used for the respective animal models.
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Lipsitz YY, Zandstra PW. Human pluripotent stem cell process parameter optimization in a small scale suspension bioreactor. BMC Proc 2015. [PMCID: PMC4685349 DOI: 10.1186/1753-6561-9-s9-o10] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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Human cardiomyocyte generation from pluripotent stem cells: A state-of-art. Life Sci 2015; 145:98-113. [PMID: 26682938 DOI: 10.1016/j.lfs.2015.12.023] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Revised: 11/08/2015] [Accepted: 12/09/2015] [Indexed: 12/11/2022]
Abstract
The human heart is considered a non-regenerative organ. Worldwide, cardiovascular diseases continue to be the leading cause of death. Despite advances in cardiac treatment, myocardial repair remains severely limited by the lack of an appropriate source of viable cardiomyocytes (CMs) to replace damaged tissue. Human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can efficiently be differentiated into functional CMs necessary for cell replacement therapy and other potential applications. The number of protocols that derive CMs from hPSCs has increased exponentially over the past decade following observation of the first human beating CMs. A number of highly efficient, chemical based protocols have been developed to generate human CMs (hCMs) in small-scale and large-scale suspension systems. To reduce the heterogeneity of hPSC-derived CMs, the differentiation protocols were modulated to exclusively generate atrial-, ventricular-, and nodal-like CM subtypes. Recently, remarkable advances have been achieved in hCM generation including chemical-based cardiac differentiation, cardiac subtype specification, large-scale suspension culture differentiation, and development of chemically defined culture conditions. These hCMs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues are overcome. Herein we review recent progress in the in vitro generation of CMs and cardiac subtypes from hPSCs and discuss their potential applications and current limitations.
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50
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Fonoudi H, Ansari H, Abbasalizadeh S, Larijani MR, Kiani S, Hashemizadeh S, Zarchi AS, Bosman A, Blue GM, Pahlavan S, Perry M, Orr Y, Mayorchak Y, Vandenberg J, Talkhabi M, Winlaw DS, Harvey RP, Aghdami N, Baharvand H. A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells. Stem Cells Transl Med 2015; 4:1482-1494. [PMID: 26511653 PMCID: PMC4675501 DOI: 10.5966/sctm.2014-0275] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2014] [Accepted: 07/08/2015] [Indexed: 12/15/2022] Open
Abstract
UNLABELLED Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.
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Affiliation(s)
- Hananeh Fonoudi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia
| | - Hassan Ansari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Mehran Rezaei Larijani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Sahar Kiani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Shiva Hashemizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Ali Sharifi Zarchi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Alexis Bosman
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia
| | - Gillian M Blue
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Sara Pahlavan
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Matthew Perry
- St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia
| | - Yishay Orr
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
| | - Yaroslav Mayorchak
- The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
| | - Jamie Vandenberg
- St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia
| | - Mahmood Talkhabi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - David S Winlaw
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Richard P Harvey
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, New South Wales, Australia
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research, Tehran, Iran
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