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De Sousa PA, Perfect L, Ye J, Samuels K, Piotrowska E, Gordon M, Mate R, Abranches E, Wishart TM, Dockrell DH, Courtney A. Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture. Stem Cell Res Ther 2024; 15:130. [PMID: 38702837 PMCID: PMC11069290 DOI: 10.1186/s13287-024-03719-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 04/05/2024] [Indexed: 05/06/2024] Open
Abstract
BACKGROUND Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.
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Affiliation(s)
- Paul A De Sousa
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK.
- Stroma Therapeutics Ltd, Glasgow, UK.
| | - Leo Perfect
- Biotherapeutics and Advanced Therapies, Science Research and Innovation Group, UK Stem Cell Bank, MHRA, South Mimms, UK
| | - Jinpei Ye
- Institute of Biomedical Science, Shanxi University, Taiyuan, Shanxi, China
| | - Kay Samuels
- Scottish National Blood Transfusion Service, Edinburgh, UK
| | - Ewa Piotrowska
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK
- Department of Molecular Biology, University of Gdansk, Gdańsk, Poland
| | - Martin Gordon
- Biotherapeutics and Advanced Therapies, Science Research and Innovation Group, UK Stem Cell Bank, MHRA, South Mimms, UK
| | - Ryan Mate
- Biotherapeutics and Advanced Therapies, Science Research and Innovation Group, UK Stem Cell Bank, MHRA, South Mimms, UK
| | - Elsa Abranches
- Biotherapeutics and Advanced Therapies, Science Research and Innovation Group, UK Stem Cell Bank, MHRA, South Mimms, UK
| | | | - David H Dockrell
- Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
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Nogueira IPM, Costa GMJ, Lacerda SMDSN. Avian iPSC Derivation to Recover Threatened Wild Species: A Comprehensive Review in Light of Well-Established Protocols. Animals (Basel) 2024; 14:220. [PMID: 38254390 PMCID: PMC10812705 DOI: 10.3390/ani14020220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Revised: 12/20/2023] [Accepted: 12/21/2023] [Indexed: 01/24/2024] Open
Abstract
Induced pluripotent stem cells (iPSCs) were first generated by Yamanaka in 2006, revolutionizing research by overcoming limitations imposed by the use of embryonic stem cells. In terms of the conservation of endangered species, iPSC technology presents itself as a viable alternative for the manipulation of target genetics without compromising specimens. Although iPSCs have been successfully generated for various species, their application in nonmammalian species, particularly avian species, requires further in-depth investigation to cover the diversity of wild species at risk and their different protocol requirements. This study aims to provide an overview of the workflow for iPSC induction, comparing well-established protocols in humans and mice with the limited information available for avian species. Here, we discuss the somatic cell sources to be reprogrammed, genetic factors, delivery methods, enhancers, a brief history of achievements in avian iPSC derivation, the main approaches for iPSC characterization, and the future perspectives and challenges for the field. By examining the current protocols and state-of-the-art techniques employed in iPSC generation, we seek to contribute to the development of efficient and species-specific iPSC methodologies for at-risk avian species. The advancement of iPSC technology holds great promise for achieving in vitro germline competency and, consequently, addressing reproductive challenges in endangered species, providing valuable tools for basic research, bird genetic preservation and rescue, and the establishment of cryobanks for future conservation efforts.
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Affiliation(s)
| | | | - Samyra Maria dos Santos Nassif Lacerda
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (I.P.M.N.); (G.M.J.C.)
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Guidotti G, Duelen R, Bloise N, Soccio M, Gazzano M, Aluigi A, Visai L, Sampaolesi M, Lotti N. The ad hoc chemical design of random PBS-based copolymers influences the activation of cardiac differentiation while altering the HYPPO pathway target genes in hiPSCs. BIOMATERIALS ADVANCES 2023; 154:213583. [PMID: 37604040 DOI: 10.1016/j.bioadv.2023.213583] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/22/2023] [Revised: 07/23/2023] [Accepted: 08/07/2023] [Indexed: 08/23/2023]
Abstract
Cardiac tissue engineering is a cutting-edge technology aiming to replace irreversibly damaged cardiac tissue and restore contractile functionality. However, cardiac tissue engineering porous and perfusable scaffolds to enable oxygen supply in vitro and eventually promote angiogenesis in vivo are still desirable. Two fully-aliphatic random copolymers of poly(butylene succinate) (PBS), poly(butylene succinate/Pripol), P(BSBPripol), and poly(butylene/neopentyl glycol succinate), P(BSNS), containing two different subunits, neopentyl glycol and Pripol 1009, were successfully synthesized and then electrospun in tridimentional fibrous mats. The copolymers show different thermal and mechanical behaviours as result of their chemical structure. In particular, copolymerization led to a reduction in crystallinity and consequently PBS stiffness, reaching values of elastic modulus very close to those of soft tissues. Then, to check the biological suitability, human induced Pluripotent Stem Cells (hiPSCs) were directly seeded on both PBS-based copolymeric scaffolds. The results confirmed the ability of both the scaffolds to sustain cell viability and to maintain their stemness during cell expansion. Furthermore, gene expression and immunofluorescence analysis showed that P(BSBPripol) scaffold promoted an upregulation of the early cardiac progenitor and later-stage markers with a simultaneously upregulation of HYPPO pathway gene expression, crucial for mechanosensing of cardiac progenitor cells. These results suggest that the correct ad-hoc chemical design and, in turn, the mechanical properties of the matrix, such as substrate stiffness, together with surface porosity, play a critical role in regulating the behaviour of cardiac progenitors, which ultimately offers valuable insights into the development of novel bio-inspired scaffolds for cardiac tissue regeneration.
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Affiliation(s)
- Giulia Guidotti
- Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy
| | - Robin Duelen
- Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium
| | - Nora Bloise
- Department of Molecular Medicine, Centre for Health Technologies (CHT), INSTM UdR of Pavia, University of Pavia, Viale Taramelli 3/B, 27100 Pavia, Italy; Medicina Clinica-Specialistica, UOR5 Laboratorio di Nanotecnologie, ICS Maugeri, IRCCS, Via Salvatore Maugeri 4, 27100 Pavia, Italy
| | - Michelina Soccio
- Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy
| | - Massimo Gazzano
- Organic Synthesis and Photoreactivity Institute, CNR, Via Gobetti 101, 40129 Bologna, Italy
| | - Annalisa Aluigi
- Department of Biomolecular Sciences, University of Urbino Carlo Bo, Piazza del Rinascimento, 6, 61029 Urbino, (PU), Italy
| | - Livia Visai
- Department of Molecular Medicine, Centre for Health Technologies (CHT), INSTM UdR of Pavia, University of Pavia, Viale Taramelli 3/B, 27100 Pavia, Italy; Medicina Clinica-Specialistica, UOR5 Laboratorio di Nanotecnologie, ICS Maugeri, IRCCS, Via Salvatore Maugeri 4, 27100 Pavia, Italy
| | - Maurilio Sampaolesi
- Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; Histology and Medical Embryology Unit, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Rome, Italy.
| | - Nadia Lotti
- Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy.
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Lin CY, Ching YY, Wu SF, Lee YK, Fan HC, Su LY, Tsai SY, Chen YC, Shen CI, Su HL. Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells. Cell Transplant 2023; 32:9636897231198172. [PMID: 37698258 PMCID: PMC10498698 DOI: 10.1177/09636897231198172] [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: 05/01/2023] [Revised: 08/11/2023] [Accepted: 08/15/2023] [Indexed: 09/13/2023] Open
Abstract
Cell expansion of human pluripotent stem cells (hPSCs) commonly depends on Matrigel as a coating matrix on two-dimensional (2D) culture plates and 3D microcarriers. However, the xenogenic Matrigel requires sophisticated quality-assurance processes to meet clinical requirements. In this study, we develop an innovative coating-free medium for expanding hPSCs. The xenofree medium supports the weekend-free culture and competitive growth of hPSCs on several cell culture plastics without an additional pre-coating process. The pluripotent stemness of the expanded cells is stably sustained for more than 10 passages, featured with high pluripotent marker expressions, normal karyotyping, and differentiating capacity for three germ layers. The expression levels of some integrins are reduced, compared with those of the hPSCs on Matrigel. This medium also successfully supports the clonal expansion and induced pluripotent stem cell establishment from mitochondrial-defective MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) patient's peripheral blood mononuclear cells. This innovative hPSC medium provides a straightforward scale-up process for producing clinical-orientated hPSCs by excluding the conventional coating procedure.
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Affiliation(s)
- Chih-Yao Lin
- Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
| | - Yu-Yun Ching
- Duogenic StemCells Corporation, Taichung, Taiwan
| | - Shih-Fang Wu
- The Joint Program of Tissue Engineering and Regenerative Medicine, National Chung Hsing University and National Health Research Institutes, Taichung, Taiwan
| | - Yi-Ko Lee
- Duogenic StemCells Corporation, Taichung, Taiwan
| | - Hueng-Chuen Fan
- Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Wuchi, Taichung, Taiwan
- Department of Nursing, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan
| | - Liang-Yu Su
- Department of Life Science, National Taiwan University, Taipei, Taiwan
| | - Su-Yi Tsai
- Department of Life Science, National Taiwan University, Taipei, Taiwan
| | - Yu-Ching Chen
- The Joint Program of Tissue Engineering and Regenerative Medicine, National Chung Hsing University and National Health Research Institutes, Taichung, Taiwan
- Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua City, Taiwan
| | - Ching-I Shen
- Duogenic StemCells Corporation, Taichung, Taiwan
| | - Hong-Lin Su
- Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
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Zhang J, Gregorich ZR, Tao R, Kim GC, Lalit PA, Carvalho JL, Markandeya Y, Mosher DF, Palecek SP, Kamp TJ. Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin. eLife 2022; 11:e69028. [PMID: 35758861 PMCID: PMC9236614 DOI: 10.7554/elife.69028] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Accepted: 06/05/2022] [Indexed: 11/13/2022] Open
Abstract
Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation.
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Affiliation(s)
- Jianhua Zhang
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
- Stem Cell and Regenerative Medicine Center, University of Wisconsin - MadisonMadisonUnited States
| | - Zachery R Gregorich
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
| | - Ran Tao
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
| | - Gina C Kim
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
| | - Pratik A Lalit
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
| | - Juliana L Carvalho
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
- Department of Genomic Sciences and Biotechnology, University of BrasíliaBrasíliaBrazil
| | - Yogananda Markandeya
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
| | - Deane F Mosher
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
- Morgridge Institute for ResearchMadisonUnited States
- Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-MadisonMadisonUnited States
| | - Sean P Palecek
- Stem Cell and Regenerative Medicine Center, University of Wisconsin - MadisonMadisonUnited States
- Department of Chemical and Biological Engineering, College of Engineering, University of WisconsinMadisonUnited States
| | - Timothy J Kamp
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
- Stem Cell and Regenerative Medicine Center, University of Wisconsin - MadisonMadisonUnited States
- Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - MadisonMadisonUnited States
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6
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Kolagar TA, Farzaneh M, Nikkar N, Khoshnam SE. Human Pluripotent Stem Cells in Neurodegenerative Diseases: Potentials, Advances and Limitations. Curr Stem Cell Res Ther 2020; 15:102-110. [PMID: 31441732 DOI: 10.2174/1574888x14666190823142911] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2019] [Revised: 06/15/2019] [Accepted: 07/17/2019] [Indexed: 12/14/2022]
Abstract
Neurodegenerative diseases are progressive and uncontrolled gradual loss of motor neurons function or death of neuron cells in the central nervous system (CNS) and the mechanisms underlying their progressive nature remain elusive. There is urgent need to investigate therapeutic strategies and novel treatments for neural regeneration in disorders like Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Currently, the development and identification of pluripotent stem cells enabling the acquisition of a large number of neural cells in order to improve cell recovery after neurodegenerative disorders. Pluripotent stem cells which consist of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their ability to indefinitely self-renew and the capacity to differentiate into different types of cells. The first human ESC lines were established from donated human embryos; while, because of a limited supply of donor embryos, human ESCs derivation remains ethically and politically controversial. Hence, hiPSCs-based therapies have been shown as an effective replacement for human ESCs without embryo destruction. Compared to the invasive methods for derivation of human ESCs, human iPSCs has opened possible to reprogram patient-specific cells by defined factors and with minimally invasive procedures. Human pluripotent stem cells are a good source for cell-based research, cell replacement therapies and disease modeling. To date, hundreds of human ESC and human iPSC lines have been generated with the aim of treating various neurodegenerative diseases. In this review, we have highlighted the recent potentials, advances, and limitations of human pluripotent stem cells for the treatment of neurodegenerative disorders.
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Affiliation(s)
- Tannaz Akbari Kolagar
- Faculty of Biological Sciences, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | - Maryam Farzaneh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Negin Nikkar
- Department of Biology, Faculty of Sciences, Alzahra University, Tehran, Iran
| | - Seyed Esmaeil Khoshnam
- Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Koch L, Deiwick A, Franke A, Schwanke K, Haverich A, Zweigerdt R, Chichkov B. Laser bioprinting of human induced pluripotent stem cells—the effect of printing and biomaterials on cell survival, pluripotency, and differentiation. Biofabrication 2018; 10:035005. [DOI: 10.1088/1758-5090/aab981] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs. Sci Rep 2018; 8:5907. [PMID: 29651156 PMCID: PMC5897327 DOI: 10.1038/s41598-018-24074-y] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Accepted: 02/28/2018] [Indexed: 01/10/2023] Open
Abstract
Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.
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Abdal Dayem A, Lee S, Y. Choi H, Cho SG. The Impact of Adhesion Molecules on the In Vitro Culture and Differentiation of Stem Cells. Biotechnol J 2018; 13:1700575. [DOI: 10.1002/biot.201700575] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
Affiliation(s)
- Ahmed Abdal Dayem
- Department of Stem Cell and Regenerative Biotechnology; Incurable Disease Animal Model and Stem Cell Institute (IDASI); Konkuk University; 120 Neungdong-ro Gwangjin-gu 05029 Seoul Republic of Korea
| | - Soobin Lee
- Department of Stem Cell and Regenerative Biotechnology; Incurable Disease Animal Model and Stem Cell Institute (IDASI); Konkuk University; 120 Neungdong-ro Gwangjin-gu 05029 Seoul Republic of Korea
| | - Hye Y. Choi
- Department of Stem Cell and Regenerative Biotechnology; Incurable Disease Animal Model and Stem Cell Institute (IDASI); Konkuk University; 120 Neungdong-ro Gwangjin-gu 05029 Seoul Republic of Korea
| | - Ssang-Goo Cho
- Department of Stem Cell and Regenerative Biotechnology; Incurable Disease Animal Model and Stem Cell Institute (IDASI); Konkuk University; 120 Neungdong-ro Gwangjin-gu 05029 Seoul Republic of Korea
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Long-Term Maintenance of Human Pluripotent Stem Cells on cRGDfK-Presenting Synthetic Surfaces. Sci Rep 2018; 8:701. [PMID: 29335618 PMCID: PMC5768753 DOI: 10.1038/s41598-018-19209-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Accepted: 12/27/2017] [Indexed: 12/12/2022] Open
Abstract
Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.
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Wong CW, Chen YT, Chien CL, Yu TY, Rwei SP, Hsu SH. A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2018; 82:69-79. [DOI: 10.1016/j.msec.2017.08.050] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/24/2017] [Revised: 07/21/2017] [Accepted: 08/10/2017] [Indexed: 10/19/2022]
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Green DW, Watson GS, Watson JA, Lee JM, Jung HS. Use of Tethered Hydrogel Microcoatings for Mesenchymal Stem Cell Equilibrium, Differentiation, and Self-Organization into Microtissues. ACTA ACUST UNITED AC 2017; 1:e1700116. [PMID: 32646160 DOI: 10.1002/adbi.201700116] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Revised: 09/08/2017] [Indexed: 01/29/2023]
Abstract
Therapeutic adult mesenchymal stem cells (MSCs) lose multipotency and multilineage specialization in culture and after transplantation due to the absence of complex biological architecture. Here, it is shown that a transient ultrathin covering of permeable biomaterial can be differentially formulated to either preserve multipotency or induce multidifferentiation. Accordingly, populations of single, spherical MSCs in suspended media with high selectivity and specificity can be coated. Assembly of single, double, and triple hydrogel layers at MSC membranes is initiated by first attaching MSC-specific immunoglobulins onto CD90 or Stro-1 receptors and UEA-1 and soybean lectins. A secondary biotinylated immunoglobulin is targeted for avidin binding, which becomes an attractor for biotinylated alginate or hyaluronate, which are subsequently stiffened and gelled, in situ around the entire cell surface. Alginate microcoatings permeated with mobile BMP-2-induced osteospecialized tissue, vascular endothelial growth factor (VEGF) induced microcapillary formation, while microcoatings, with selected basement membrane proteins, preserve the multipotent phenotype of MSCs, for continuing rounds of culture and directed specialization. Furthermore, forced packing of microcoated MSC populations creates prototypical tissue compartments: the coating partially simulating the extracellular matrix structures. Remarkably, microcoated MSC clusters show a tremendous simulation of a common embryological tissue transformation into the epithelium. Thus, confinement of free morphology exerts another control on tissue specialization and formation.
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Affiliation(s)
- David W Green
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea.,Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Sai Ying Pun, Hong Kong, SAR
| | - Gregory S Watson
- School of Science and Engineering, University of the Sunshine Coast, Hervey Bay, QLD, 4655, Australia
| | - Jolanta A Watson
- School of Science and Engineering, University of the Sunshine Coast, Hervey Bay, QLD, 4655, Australia
| | - Jong-Min Lee
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea
| | - Han-Sung Jung
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea.,Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Sai Ying Pun, Hong Kong, SAR
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13
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Lu J, Kaestle K, Huang J, Liu Q, Zhang P, Gao L, Gardiner J, Thissen H, Yang HT. Interactions of human embryonic stem cell-derived cardiovascular progenitor cells with immobilized extracellular matrix proteins. J Biomed Mater Res A 2017; 105:1094-1104. [PMID: 28085215 DOI: 10.1002/jbm.a.36005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 12/19/2016] [Accepted: 01/10/2017] [Indexed: 11/11/2022]
Abstract
Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However, little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm), fibronectin (Fn), collagen I (CI), collagen III (CIII), and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI, CIII, CIV, and Fn and their combinations were important for hESC-CVPC attachment and survival, while Lm was dispensable. Moreover, for coatings displaying single ECM proteins, CI and CIII performed better than CIV and Fn, while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1094-1104, 2017.
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Affiliation(s)
- Jizhen Lu
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
| | - Katrin Kaestle
- CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria, 3168, Australia
| | - Jijun Huang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
| | - Qiao Liu
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
| | - Peng Zhang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
| | - Ling Gao
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
| | - James Gardiner
- CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria, 3168, Australia
| | - Helmut Thissen
- CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria, 3168, Australia
| | - Huang-Tian Yang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Biological Research Building A, 320 Yueyang Road, Shanghai 200031, China.,Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China
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14
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Walczak MP, Drozd AM, Stoczynska-Fidelus E, Rieske P, Grzela DP. Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors. J Transl Med 2016; 14:341. [PMID: 27998294 PMCID: PMC5168869 DOI: 10.1186/s12967-016-1097-0] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2016] [Accepted: 11/24/2016] [Indexed: 12/21/2022] Open
Abstract
Background Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. Methods We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. Results Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. Conclusions Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1097-0) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Maciej P Walczak
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland
| | - Anna M Drozd
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland
| | - Ewelina Stoczynska-Fidelus
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.,Department of Tumor Biology, Medical University of Łódź, Żeligowskiego 7/9, 90-752, Łódź, Poland
| | - Piotr Rieske
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.,Department of Tumor Biology, Medical University of Łódź, Żeligowskiego 7/9, 90-752, Łódź, Poland.,Research and Development Unit, Personather Ltd., Milionowa 23, 93-193, Łódź, Poland
| | - Dawid P Grzela
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.
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15
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Wang PY, Thissen H, Kingshott P. Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review. Acta Biomater 2016; 45:31-59. [PMID: 27596488 DOI: 10.1016/j.actbio.2016.08.054] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Revised: 07/30/2016] [Accepted: 08/30/2016] [Indexed: 02/08/2023]
Abstract
The ability to control the interactions of stem cells with synthetic surfaces is proving to be effective and essential for the quality of passaged stem cells and ultimately the success of regenerative medicine. The stem cell niche is crucial for stem cell self-renewal and differentiation. Thus, mimicking the stem cell niche, and here in particular the extracellular matrix (ECM), in vitro is an important goal for the expansion of stem cells and their applications. Here, surface nanotopographies and surface-immobilised biosignals have been identified as major factors that control stem cell responses. The development of tailored surfaces having an optimum nanotopography and displaying suitable biosignals is proposed to be essential for future stem cell culture, cell therapy and regenerative medicine applications. While early research in the field has been restricted by the limited availability of micro- and nanofabrication techniques, new approaches involving the use of advanced fabrication and surface immobilisation methods are starting to emerge. In addition, new cell types such as induced pluripotent stem cells (iPSCs) have become available in the last decade, but have not been fully understood. This review summarises significant advances in the area and focuses on the approaches that are aimed at controlling the behavior of human stem cells including maintenance of their self-renewal ability and improvement of their lineage commitment using nanotopographies and biosignals. More specifically, we discuss developments in biointerface science that are an important driving force for new biomedical materials and advances in bioengineering aiming at improving stem cell culture protocols and 3D scaffolds for clinical applications. Cellular responses revolve around the interplay between the surface properties of the cell culture substrate and the biomolecular composition of the cell culture medium. Determination of the precise role played by each factor, as well as the synergistic effects amongst the factors, all of which influence stem cell responses is essential for future developments. This review provides an overview of the current state-of-the-art in the design of complex material surfaces aimed at being the next generation of tools tailored for applications in cell culture and regenerative medicine. STATEMENT OF SIGNIFICANCE This review focuses on the effect of surface nanotopographies and surface-bound biosignals on human stem cells. Recently, stem cell research attracts much attention especially the induced pluripotent stem cells (iPSCs) and direct lineage reprogramming. The fast advance of stem cell research benefits disease treatment and cell therapy. On the other hand, surface property of cell adhered materials has been demonstrated very important for in vitro cell culture and regenerative medicine. Modulation of cell behavior using surfaces is costeffective and more defined. Thus, we summarise the recent progress of modulation of human stem cells using surface science. We believe that this review will capture a broad audience interested in topographical and chemical patterning aimed at understanding complex cellular responses to biomaterials.
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16
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Dai S, Li R, Long Y, Titus S, Zhao J, Huang R, Xia M, Zheng W. One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays. ACTA ACUST UNITED AC 2016; 21:1112-1124. [PMID: 27647668 DOI: 10.1177/1087057116670068] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate precoating, hampering its applications in high-throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate precoating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high-throughput assays using neuronal cells differentiated from human stem cells for translational research.
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Affiliation(s)
- Sheng Dai
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.,2 Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Rong Li
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Yan Long
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.,3 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Steve Titus
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Jinghua Zhao
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Ruili Huang
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Menghang Xia
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Wei Zheng
- 1 National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
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17
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Badenes SM, Fernandes TG, Cordeiro CSM, Boucher S, Kuninger D, Vemuri MC, Diogo MM, Cabral JMS. Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems. PLoS One 2016; 11:e0151264. [PMID: 26999816 PMCID: PMC4801338 DOI: 10.1371/journal.pone.0151264] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2015] [Accepted: 02/19/2016] [Indexed: 12/24/2022] Open
Abstract
Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
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Affiliation(s)
- Sara M. Badenes
- Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Tiago G. Fernandes
- Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- * E-mail:
| | - Cláudia S. M. Cordeiro
- Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Shayne Boucher
- Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America
| | - David Kuninger
- Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America
| | - Mohan C. Vemuri
- Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America
| | - Maria Margarida Diogo
- Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America
| | - Joaquim M. S. Cabral
- Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America
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18
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Lee H, Nam D, Choi JK, Araúzo-Bravo MJ, Kwon SY, Zaehres H, Lee T, Park CY, Kang HW, Schöler HR, Kim JB. Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene. Sci Rep 2016; 6:20708. [PMID: 26846167 PMCID: PMC4742916 DOI: 10.1038/srep20708] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2015] [Accepted: 01/11/2016] [Indexed: 12/12/2022] Open
Abstract
The maintenance of undifferentiated human pluripotent stem cells (hPSC) under
xeno-free condition requires the use of human feeder cells or extracellular matrix
(ECM) coating. However, human-derived sources may cause human pathogen contamination
by viral or non-viral agents to the patients. Here we demonstrate feeder-free and
xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown
nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which
completely rule out the concern of human pathogen contamination. DAS-NG exhibited
advanced biocompatibilities including surface nanoroughness, oxygen containing
functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain
pluripotency in vitro and in vivo, and especially cell
adhesion-related gene expression profile was comparable to those of cultured on
feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high
expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free
culture method using DAS-NG will facilitate the generation of clinical-grade
hPSC.
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Affiliation(s)
- Hyunah Lee
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Donggyu Nam
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Jae-Kyung Choi
- SMEs Support Center, Korea Institute of Science and Technology Information, 48058 Busan, South Korea
| | - Marcos J Araúzo-Bravo
- Group of Computational Biology and Bioinformatics, Biodonostia Health Research Institute, 20014 San Sebastián, Spain.,IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
| | - Soon-Yong Kwon
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea.,School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Holm Zaehres
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany
| | - Taehee Lee
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Chan Young Park
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Hyun-Wook Kang
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
| | - Hans R Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany
| | - Jeong Beom Kim
- Hans Schöler Stem Cell Research Center (HSSCRC), School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 44919 Ulsan, South Korea
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19
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Abstract
Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in the human body, they reside in specialized microenvironments called "stem cell niches." Although several niches have been described and studied in vivo, their functional replication in vitro is still incomplete. The in vitro culture of pluripotent stem cells may represent one of the most advanced examples in the effort to create an artificial or synthetic stem cell niche. A focus has been placed on the development of human stem cell microenvironments due to their significant clinical implications, in addition to the potential differences between animal and human cells. In this concise review we describe the advances in human pluripotent stem cell culture, and explore the idea that the knowledge gained from this model could be replicated to create synthetic niches for other human stem cell populations, which have proven difficult to maintain in vitro.
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20
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Kopaczka K, Skowron K, Kolanko E, Czekaj P. The relationship between amniotic epithelial cells and their microenvironment. J Appl Biomed 2016. [DOI: 10.1016/j.jab.2015.10.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
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21
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Zhou Y, Mao H, Joddar B, Umeki N, Sako Y, Wada KI, Nishioka C, Takahashi E, Wang Y, Ito Y. The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness. Sci Rep 2015; 5:11386. [PMID: 26065582 PMCID: PMC4464345 DOI: 10.1038/srep11386] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2014] [Accepted: 04/22/2015] [Indexed: 11/09/2022] Open
Abstract
The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs. Biological activity was monitored by colony formation of iPS cells. Treatment with a low concentration of FA sustained the membrane fluidity and biological activity, which were comparable to those of mitomycin C-treated MEFs. The biological activity was further confirmed by sustained expression of alkaline phosphatase, SSEA-1, and other pluripotency markers in iPS cells after 3-5 days of culture on FA-fixed MEFs. Chemical fixation of feeder cells has several advantages such as providing ready-to-use culture substrates without contamination by proliferating feeder cells. Therefore, our results provide an important basis for the development of chemically fixed culture substrates for pluripotent stem cell culture as an alternative to conventional treatment by mitomycin C or x-ray irradiation.
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Affiliation(s)
- Yue Zhou
- Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
- School of Nursing, Nanjing University of Chinese Medicine, 138 Xianlin Road, Qixia District, Nanjing, Jiangsu Province 210023, China
- Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, No.1266 Fujin Road, Changchun 130021, China
| | - Hongli Mao
- Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Binata Joddar
- Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Nobuhisa Umeki
- Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Yasushi Sako
- Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Ken-Ichi Wada
- Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Chieko Nishioka
- Support Unit for Animal Experiment, Research Resources Center, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Eiki Takahashi
- Support Unit for Animal Experiment, Research Resources Center, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Yi Wang
- Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, No.1266 Fujin Road, Changchun 130021, China
| | - Yoshihiro Ito
- Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
- Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1Hirosawa, Wako, Saitama 351-0198, Japan
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22
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Silva MM, Rodrigues AF, Correia C, Sousa MFQ, Brito C, Coroadinha AS, Serra M, Alves PM. Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization. Stem Cells Transl Med 2015; 4:731-42. [PMID: 25979863 DOI: 10.5966/sctm.2014-0270] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 03/09/2015] [Indexed: 12/30/2022] Open
Abstract
UNLABELLED : Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems. SIGNIFICANCE The clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in well-defined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.
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Affiliation(s)
- Marta M Silva
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Ana F Rodrigues
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Cláudia Correia
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Marcos F Q Sousa
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Ana S Coroadinha
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Margarida Serra
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Paula M Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
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23
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Fonseca SAS, Costas RM, Pereira LV. Searching for naïve human pluripotent stem cells. World J Stem Cells 2015; 7:649-656. [PMID: 25914771 PMCID: PMC4404399 DOI: 10.4252/wjsc.v7.i3.649] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2014] [Accepted: 01/20/2015] [Indexed: 02/06/2023] Open
Abstract
Normal mouse pluripotent stem cells were originally derived from the inner cell mass (ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells (ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICM-derived epiblast of post-implantation embryos. These mouse epiblast stem cells (EpiSCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into EpiSC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called naïve (or ground state) human ESCs, and the derivation of naïve human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how (and whether) these relate to different stages of embryonic development, and discuss the potential implications of naïve human ESCs in research and therapy.
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Dang LTH, Feric NT, Laschinger C, Chang WY, Zhang B, Wood GA, Stanford WL, Radisic M. Inhibition of apoptosis in human induced pluripotent stem cells during expansion in a defined culture using angiopoietin-1 derived peptide QHREDGS. Biomaterials 2014; 35:7786-99. [PMID: 24930852 DOI: 10.1016/j.biomaterials.2014.05.018] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2014] [Accepted: 05/08/2014] [Indexed: 02/07/2023]
Abstract
Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival, self-renewal, and differentiation. Thus, hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel, i.e. on standard substrates, without affecting hPSC morphology, growth rate or the ability to differentiate into multiple lineages both in vitro and in vivo. Importantly, QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically, the interaction of QHREDGS with β1-integrins increased expression of integrin-linked kinase (ILK), increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2), and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance.
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Affiliation(s)
- Lan T H Dang
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; The Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Nicole T Feric
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada
| | - Carol Laschinger
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada
| | - Wing Y Chang
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; Sprott Centre for Stem Cell Research, The Ottawa Hospital Research Institute, Ottawa, Ontario K1H 8L6, Canada; Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
| | - Boyang Zhang
- Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada
| | - Geoffrey A Wood
- Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
| | - William L Stanford
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; Sprott Centre for Stem Cell Research, The Ottawa Hospital Research Institute, Ottawa, Ontario K1H 8L6, Canada; Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
| | - Milica Radisic
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; The Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
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Joddar B, Hoshiba T, Chen G, Ito Y. Stem cell culture using cell-derived substrates. Biomater Sci 2014; 2:1595-1603. [DOI: 10.1039/c4bm00126e] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
There have been great efforts to develop cell culture systems using chemically-fixed cells or decellularized matrices to regulate stem cell functions.
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Affiliation(s)
| | - Takashi Hoshiba
- Department of Biochemical Engineering
- Graduate School of Science and Engineering
- Yamagata University
- Yonezawa, Japan
- Tissue Regeneration Materials Unit
| | - Guoping Chen
- Tissue Regeneration Materials Unit
- International Center for Materials Nanoarchitectonics
- National Institute for Materials Science
- Tsukuba, Japan
| | - Yoshihiro Ito
- Nano Medical Engineering Laboratory
- RIKEN
- Wako, Japan
- Emergent Bioengineering Materials Research Team
- RIKEN Center for Emergent Matter Science
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