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Karami N, Taei A, Eftekhari-Yazdi P, Hassani F. Signaling pathway regulators in preimplantation embryos. J Mol Histol 2024; 56:57. [PMID: 39729177 DOI: 10.1007/s10735-024-10338-7] [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: 08/31/2024] [Accepted: 12/12/2024] [Indexed: 12/28/2024]
Abstract
Embryonic development during the preimplantation stages is highly sensitive and critically dependent on the reception of signaling cues. The precise coordination of diverse pathways and signaling factors is essential for successful embryonic progression. Even minor disruptions in these factors can result in physiological dysfunction, fetal malformations, or embryonic arrest. This issue is particularly evident in assisted reproductive technologies, such as in vitro fertilization, where embryonic arrest is frequently observed. A detailed understanding of these pathways enhances insight into the fundamental mechanisms underlying cellular processes and their contributions to embryonic development. The significance of elucidating signaling pathways and their regulatory factors in preimplantation development cannot be overstated. The application of this knowledge in laboratory settings has the potential to support strategies for modeling developmental stages and diseases, drug screening, therapeutic discovery, and reducing embryonic arrest. Furthermore, using various factors, small molecules, and pharmacological agents can enable the development or optimization of culture media for enhanced embryonic viability. While numerous pathways influence preimplantation development, this study examines several critical signaling pathways in this contex.
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Affiliation(s)
- Narges Karami
- MSc., Faculty of Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran
| | - Adeleh Taei
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Poopak Eftekhari-Yazdi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, P.O.Box 16635-148, Tehran, Iran
| | - Fatemeh Hassani
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, P.O.Box 16635-148, Tehran, Iran.
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Wang T, Yu T, Tsai CY, Hong ZY, Chao WH, Su YS, Subbiah SK, Renuka RR, Hsu ST, Wu GJ, Higuchi A. Xeno-free culture and proliferation of hPSCs on 2D biomaterials. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 199:63-107. [PMID: 37678982 DOI: 10.1016/bs.pmbts.2023.02.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/16/2023]
Abstract
Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.
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Affiliation(s)
- Ting Wang
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Tao Yu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Chang-Yen Tsai
- Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan
| | - Zhao-Yu Hong
- Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan
| | - Wen-Hui Chao
- Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan
| | - Yi-Shuo Su
- Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan
| | - Suresh Kumar Subbiah
- Centre for Materials Engineering and Regenerative Medicine, Bharath Institute of Higher Education and Research, Chennai, India
| | - Remya Rajan Renuka
- Centre for Materials Engineering and Regenerative Medicine, Bharath Institute of Higher Education and Research, Chennai, India
| | - Shih-Tien Hsu
- Department of Internal Medicine, Landseed International Hospital, Pingjen City, Taoyuan, Taiwan
| | - Gwo-Jang Wu
- Graduate Institute of Medical Sciences and Department of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
| | - Akon Higuchi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China; Graduate Institute of Medical Sciences and Department of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
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Alsobaie S, Alsobaie T, Mantalaris S. Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells. STEM CELLS AND CLONING: ADVANCES AND APPLICATIONS 2022; 15:43-52. [PMID: 35812359 PMCID: PMC9259205 DOI: 10.2147/sccaa.s365776] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 06/22/2022] [Indexed: 01/16/2023]
Abstract
Introduction High-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application. Methods To address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells. Results Through immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities. Discussion We showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.
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Affiliation(s)
- Sarah Alsobaie
- Department of Clinical Laboratory Science, King Saud University, Riyadh, 11451, Saudi Arabia
- Correspondence: Sarah Alsobaie, Department of Clinical Laboratory Science, King Saud University, Prince Turki Alawal Street, Riyadh, 11451, Saudi Arabia, Tel +966 507191011, Fax +966 114677580, Email
| | - Tamador Alsobaie
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, SW7 2AZ, UK
| | - Sakis Mantalaris
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30322, USA
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Soroushzadeh S, Karamali F, Masaeli E, Atefi A, Nasr Esfahani MH. Scaffold free retinal pigment epithelium sheet engineering using modified alginate-RGD hydrogel. J Biosci Bioeng 2022; 133:579-586. [PMID: 35339352 DOI: 10.1016/j.jbiosc.2022.02.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 01/30/2022] [Accepted: 02/02/2022] [Indexed: 11/16/2022]
Abstract
Tissue-specific extracellular matrix (ECM) plays a critical role in cell survival and homeostasis, which are particularly essential for directing differentiation of different complex tissues such as retina. However, ECM maintenance should be considered to design an effective therapeutic strategy for retina regeneration. To achieve this, cell sheet engineering has emerged as a growing approach to closely reconstruct basal membrane of cells through a scaffold-free manner. Several irreversible sight-threatening diseases are characterized by the dysfunction and lose of retinal pigment epithelium (RPE), leading to vision loss and eventually total blindness in patients. According to impressive developments in achievement of RPE from human embryonic stem cells (hESCs), we obtained RPE cells without any extrinsic factors in a co-culture system, and cultured them on a temporary alginate hydrogel substrate. Subsequently, Arg-Gly-Asp (RGD) peptide was superficially immobilized on the upper layer of hydrogel to improve cell attachment before harvesting sheet layer. RPE cell sheet layer was released by treating pre-seeded hydrogels with sodium citrate as a calcium chelating agent and characterized in both in vitro and in vivo models. RPE sheets formed tight junction and expressed high levels of retina structural markers such as ZO-1, Bestrophin and Collagen type IV. One week after in vivo transplantation of RPE sheet, cells survived in the subretinal space, indicating that our harvesting method is non-invasive. To sum up, we introduced a unique scaffold-free method for RPE cell sheet engineering, which can find potential use for future therapeutic purposes.
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Affiliation(s)
- Sareh Soroushzadeh
- ACECR Institute of Higher Education (Isfahan Branch), P.O. Box: 84175443, Iran; Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O. Box 8159358686, Iran
| | - Fereshteh Karamali
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O. Box 8159358686, Iran
| | - Elahe Masaeli
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O. Box 8159358686, Iran
| | - Atefeh Atefi
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O. Box 8159358686, Iran
| | - Mohammad Hossein Nasr Esfahani
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O. Box 8159358686, Iran.
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Uhrig M, Ezquer F, Ezquer M. Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells. Cells 2022; 11:799. [PMID: 35269421 PMCID: PMC8909336 DOI: 10.3390/cells11050799] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 02/11/2022] [Accepted: 02/16/2022] [Indexed: 02/04/2023] Open
Abstract
Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.
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Affiliation(s)
- Markus Uhrig
- Center for Regenerative Medicine, School of Medicine, Clínica Alemana-Universidad del Desarrollo, Santiago 7610658, Chile;
| | | | - Marcelo Ezquer
- Center for Regenerative Medicine, School of Medicine, Clínica Alemana-Universidad del Desarrollo, Santiago 7610658, Chile;
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Savoj S, Esfahani MHN, Karimi A, Karamali F. Integrated stem cells from apical papilla in a 3D culture system improve human embryonic stem cell derived retinal organoid formation. Life Sci 2022; 291:120273. [PMID: 35016877 DOI: 10.1016/j.lfs.2021.120273] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 12/15/2021] [Accepted: 12/23/2021] [Indexed: 01/08/2023]
Abstract
AIM Eye organoids are 3D models of the retina that provide new possibilities for studying retinal development, drug toxicity and the molecular mechanisms of diseases. Although there are several protocols that can be used to generate functional tissues, none have been used to assemble human retinal organoids containing mesenchymal stem cells (MSCs). MAIN METHODS In this study we intend to assess the effective interactions of MSCs and human embryonic stem cells (hESCs) during retinal organoid formation. We evaluated the inducing activities of bone marrow MSCs (BM-MSCs), trabecular meshwork (TM), and stem cells from apical papilla (SCAP)-derived MSCs in differentiation of hESCs in a three-dimensional (3D) direct co-culture system. KEY FINDINGS In comparison with the two other MSC sources, the induction potential of SCAP was confirmed in the co-culture system. Although the different SCAP cell ratios did not show any significant morphology changes during the first seven days, increasing the number of SCAPs improved formation of the optic vesicle (OV) structure, which was confirmed by assessment of specific markers. The OVs subsequently developed to an optic cup (OC), which was similar to the in vivo environment. These arrangements expressed MITF in the outer layer and CHX10 in the inner layer. SIGNIFICANCE We assessed the inducing activity of SCAP during differentiation of hESCs towards a retinal fate in a 3D organoid system. However, future studies be conducted to gather additional details about the development of the eye field, retinal differentiation, and the molecular mechanisms of diseases.
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Affiliation(s)
- Soraya Savoj
- Department of Biology, University of Payam Noor, Isfahan, Iran; Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
| | - Mohammad Hossein Nasr Esfahani
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
| | - Akbar Karimi
- Department of Biology, University of Payam Noor, Isfahan, Iran.
| | - Fereshteh Karamali
- Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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8
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Meiser I, Majer J, Katsen-Globa A, Schulz A, Schmidt K, Stracke F, Koutsouraki E, Witt G, Keminer O, Pless O, Gardner J, Claussen C, Gribbon P, Neubauer JC, Zimmermann H. Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity. Cryobiology 2021; 103:57-69. [PMID: 34582849 DOI: 10.1016/j.cryobiol.2021.09.010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 09/22/2021] [Accepted: 09/23/2021] [Indexed: 10/20/2022]
Abstract
The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.
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Affiliation(s)
- Ina Meiser
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany.
| | - Julia Majer
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany
| | - Alisa Katsen-Globa
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany
| | - André Schulz
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany
| | - Katharina Schmidt
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany
| | - Frank Stracke
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany
| | | | - Gesa Witt
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, ScreeningPort, 22525, Hamburg, Germany
| | - Oliver Keminer
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, ScreeningPort, 22525, Hamburg, Germany
| | - Ole Pless
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, ScreeningPort, 22525, Hamburg, Germany
| | - John Gardner
- Censo Biotechnologies Ltd, Roslin Midlothian, EH25 9RG, United Kingdom
| | - Carsten Claussen
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, ScreeningPort, 22525, Hamburg, Germany
| | - Philip Gribbon
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, ScreeningPort, 22525, Hamburg, Germany
| | - Julia C Neubauer
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany; Fraunhofer Project Centre for Stem Cell Process Engineering, 97081, Würzburg, Germany
| | - Heiko Zimmermann
- Fraunhofer Institute for Biomedical Engineering IBMT, 66280, Sulzbach, Saar, Germany; Censo Biotechnologies Ltd, Roslin Midlothian, EH25 9RG, United Kingdom; Faculty of Marine Science, Universidad Católica Del Norte, 1781421, Coquimbo, Chile; Chair for Molecular and Cellular Biotechnology / Nanotechnology, Saarland University, 66123, Saarbrücken, Germany
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9
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Gholami S, Mazidi Z, Pahlavan S, Moslem F, Hosseini M, Taei A, Hesaraki M, Barekat M, Aghdami N, Baharvand H. A Novel Insight into Endothelial and Cardiac Cells Phenotype in Systemic Sclerosis Using Patient-Derived Induced Pluripotent Stem Cell. CELL JOURNAL 2021; 23:273-287. [PMID: 34308570 PMCID: PMC8286459 DOI: 10.22074/cellj.2021.7244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/03/2019] [Accepted: 01/26/2020] [Indexed: 12/03/2022]
Abstract
OBJECTIVE Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). MATERIALS AND METHODS In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). RESULTS SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation of SNAI1 (snail family transcriptional repressor 1) was observed in SSc-EC which might underlie VE-cadherin downregulation. Furthermore, SSc-derived CM (SSc-CM) successfully expressed cardiacspecific markers including ion channels, resulting in normal physiological behavior and responsiveness to cardioactive drugs. CONCLUSION This study provides an insight into impaired angiogenesis observed in SSc patients by evaluating in vitro cardiovascular differentiation of SSc iPSC.
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Affiliation(s)
- Sedigheh Gholami
- 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
| | - Zahra Mazidi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sara Pahlavan
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Fariba Moslem
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mahya Hosseini
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Adeleh Taei
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mahdi Hesaraki
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Maryam Barekat
- Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Nasser Aghdami
- Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - 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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10
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Gao L, Nath SC, Jiao X, Zhou R, Nishikawa S, Krawetz R, Li X, Rancourt DE. Post-Passage rock inhibition induces cytoskeletal aberrations and apoptosis in Human embryonic stem cells. Stem Cell Res 2019; 41:101641. [PMID: 31710913 DOI: 10.1016/j.scr.2019.101641] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2019] [Revised: 10/16/2019] [Accepted: 10/25/2019] [Indexed: 02/08/2023] Open
Abstract
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are prone to anoikis after single cell dissociation. The small molecule, Y-27632 is known to increase survival of hESCs and hiPSCs by inhibiting the Rho-associated protein kinase (ROCK). However, the underlying mechanisms are still unclear. Here, we thoroughly screened small molecules to investigate the adhesion and survival of hESCs in adherent culture. Y-27632 provided higher adhesion and survival of hESCs by increased cell migration and preventing cell blebbing in single dissociated cells. The combination of Matrigel with poly-d-lysine increased the attachment and survival of dissociated cells via actin filament and microtubule spreading in Y-27632-treated cells. Although Y-27632 prevented apoptosis by suppressing actin filament contraction, microtubule bundling, and blebbing, prolonged Y-27632 treatment presented a different morphology in the attached growing hESC colony. It induced apoptosis of cells by promoting cytoplasmic spread, E-cadherin structural change, and increased detachment. It also induced actin cytoskeleton disruption, combined with microtubule and intermediate filament elongation. For optimal hPSC culture, our research suggests that Y-27632 should be removed shortly after passaging.
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Affiliation(s)
- Lijie Gao
- College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071000, China
| | - Suman C Nath
- Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, T2N 4N1 Calgary, Canada; McCaig Institute for Bone and Joint Health, University of Calgary, Calgary T2N 4N1, Canada
| | - Xiyao Jiao
- College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071000, China
| | - Rongyan Zhou
- College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071000, China
| | - Sandra Nishikawa
- Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, T2N 4N1 Calgary, Canada
| | - Roman Krawetz
- McCaig Institute for Bone and Joint Health, University of Calgary, Calgary T2N 4N1, Canada
| | - Xiangyun Li
- College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071000, China.
| | - Derrick E Rancourt
- Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, T2N 4N1 Calgary, Canada; McCaig Institute for Bone and Joint Health, University of Calgary, Calgary T2N 4N1, Canada.
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11
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Okada H, Nakanishi C, Yoshida S, Shimojima M, Yokawa J, Mori M, Tada H, Yoshimuta T, Hayashi K, Yamano T, Hanayama R, Yamagishi M, Kawashiri MA. Function and Immunogenicity of Gene-corrected iPSC-derived Hepatocyte-Like Cells in Restoring Low Density Lipoprotein Uptake in Homozygous Familial Hypercholesterolemia. Sci Rep 2019; 9:4695. [PMID: 30886174 PMCID: PMC6423040 DOI: 10.1038/s41598-019-41056-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Accepted: 02/18/2019] [Indexed: 01/08/2023] Open
Abstract
Gene correction of induced pluripotent stem cells (iPSCs) has therapeutic potential for treating homozygous familial hypercholesterolemia (HoFH) associated with low-density lipoprotein (LDL) receptor (LDLR) dysfunction. However, few data exist regarding the functional recovery and immunogenicity of LDLR gene-corrected iPSC-derived hepatocyte-like cells (HLCs) obtained from an HoFH patient. Therefore, we generated iPSC-derived HLCs from an HoFH patient harbouring a point mutation (NM_000527.4:c.901 G > T) in exon 6 of LDLR, and examined their function and immunogenicity. From the patient’s iPSCs, one homozygous gene-corrected HoFH-iPSC clone and two heterozygous clones were generated using the CRISPR/Cas9 method. Both types of iPSC-derived HLCs showed recovery of the function of LDL uptake in immunofluorescence staining analysis. Furthermore, these gene-corrected iPSC-derived HLCs showed little immunogenicity against the patient’s peripheral blood mononuclear cells in a cell-mediated cytotoxicity assay. These results demonstrate that LDL uptake of iPSC-derived HLCs from HoFH can be restored by gene correction without the appearance of further immunogenicity, suggesting that gene-corrected iPSC-derived HLCs are applicable to the treatment of HoFH.
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Affiliation(s)
- Hirofumi Okada
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Chiaki Nakanishi
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Shohei Yoshida
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Masaya Shimojima
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Junichiro Yokawa
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Masayuki Mori
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Hayato Tada
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Tsuyoshi Yoshimuta
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Kenshi Hayashi
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
| | - Tomoyoshi Yamano
- Department of Immunology, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8640, Japan
| | - Rikinari Hanayama
- Department of Immunology, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8640, Japan
| | - Masakazu Yamagishi
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan. .,Department of Human Sciences, Osaka University of Human Sciences, 1-12-13 Shoya, Settsu, Osaka, 566-8501, Japan.
| | - Masa-Aki Kawashiri
- Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa, 920-8641, Japan
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12
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Kaindl J, Meiser I, Majer J, Sommer A, Krach F, Katsen-Globa A, Winkler J, Zimmermann H, Neubauer JC, Winner B. Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification. Stem Cells Transl Med 2018; 8:247-259. [PMID: 30456912 PMCID: PMC6392398 DOI: 10.1002/sctm.18-0121] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 07/31/2018] [Accepted: 08/20/2018] [Indexed: 12/22/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow‐rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post‐thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post‐thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual‐center study, we compared the results by immunocytochemistry (ICC), fluorescence‐activated cell sorting analysis, and RNA‐sequencing (RNA‐seq). Adherent vitrification was achieved in the so‐called TWIST substrate, a device combining cultivation, vitrification, storage, and post‐thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results were not significantly different at day 4 after thawing. RNA‐seq and ICC of hiPSCs revealed no change in gene expression and pluripotency markers, indicating that physical damage of slow‐rate freezing disrupts cellular membranes. Scanning electron microscopy showed preserved colony integrity by adherent vitrification. Experiments using smNPCs demonstrated that adherent vitrification is also applicable to neural derivatives of hiPSCs. Our data suggest that, compared to the state‐of‐the‐art slow‐rate freezing in suspension, adherent vitrification is an improved cryopreservation technique for hiPSCs and derivatives. stem cells translational medicine2019;8:247&259
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Affiliation(s)
- Johanna Kaindl
- Department of Stem Cell Biology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany
| | - Ina Meiser
- Fraunhofer Institute for Biomedical Engineering, Joseph-von-Fraunhofer-Weg 1, Sulzbach, Germany
| | - Julia Majer
- Fraunhofer Institute for Biomedical Engineering, Joseph-von-Fraunhofer-Weg 1, Sulzbach, Germany
| | - Annika Sommer
- Department of Stem Cell Biology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany
| | - Florian Krach
- Department of Stem Cell Biology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany.,Department of Cellular and Molecular Medicine, University of California, San Diego, California
| | - Alisa Katsen-Globa
- Fraunhofer Institute for Biomedical Engineering, Joseph-von-Fraunhofer-Weg 1, Sulzbach, Germany
| | - Jürgen Winkler
- Department of Molecular Neurology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany
| | - Heiko Zimmermann
- Fraunhofer Institute for Biomedical Engineering, Joseph-von-Fraunhofer-Weg 1, Sulzbach, Germany.,Chair for Molecular and Cellular Biotechnology/Nanotechnology, Saarland University, Saarbruecken, Germany.,Faculty of Marine Science, Universidad Católica del Norte, Coquimbo, Chile
| | - Julia C Neubauer
- Fraunhofer Institute for Biomedical Engineering, Joseph-von-Fraunhofer-Weg 1, Sulzbach, Germany.,Fraunhofer Project Centre for Stem Cell Process Engineering, Würzburg, Germany
| | - Beate Winner
- Department of Stem Cell Biology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany
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13
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Ntai A, La Spada A, De Blasio P, Biunno I. Trehalose to cryopreserve human pluripotent stem cells. Stem Cell Res 2018; 31:102-112. [PMID: 30071393 DOI: 10.1016/j.scr.2018.07.021] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2018] [Revised: 06/22/2018] [Accepted: 07/23/2018] [Indexed: 02/08/2023] Open
Abstract
The successful exploitation of human pluripotent stem cells (hPSCs) for research, translational or commercial reasons requires the implementation of a simple and efficient cryopreservation method. Cryopreservation is usually performed with dimethylsulphoxide (DMSO), in addition to animal proteins. However, even at sub-toxic levels, DMSO diminishes the pluripotency capacity of hPSCs and affects their epigenetic system by acting on the three DNA methyltransferases (Dnmts) and histone modification enzymes. Our study aimed to test trehalose-based cryosolutions containing ethylene glycol (EG) or glycerol (GLY) on hESCs RC17, hiPSCs CTR2#6 and long-term neuroepithelial-like stem cells (lt-NES) AF22. Here, we demostrate the effectiveness of these cryosolutions in hPSCs by showing an acceptable rate of cell viability and high stability compared to standard 10% DMSO freezing medium (CS10). All cell lines retained their morphology, self renewal potential and pluripotency, and none of the cryosolutions affected their differentiation potential. Genotoxicity varied among different stem cells types, while trehalose-based cryopreservation did not sensibly alter the homeostasis of endoplasmic reticulum (ER). This study provides evidence that pluripotent and neural stem cells stored in trehalose alone or with other cryoprotectants (CPAs) maintain their functional properties, indicating their potential use in cell therapies if produced in good manufacturing practice (GMP) facility.
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Affiliation(s)
- Aikaterini Ntai
- Integrated Systems Engineering S.r.l. (ISENET), Via G. Fantoli 16/15, 20138 Milan, Italy
| | - Alberto La Spada
- Institute of Genetic and Biomedical Research, National Research Council (IRGB-CNR), Via G. Fantoli 16/15, 20138 Milan, Italy
| | - Pasquale De Blasio
- Integrated Systems Engineering S.r.l. (ISENET), Via G. Fantoli 16/15, 20138 Milan, Italy.
| | - Ida Biunno
- Institute of Genetic and Biomedical Research, National Research Council (IRGB-CNR), Via G. Fantoli 16/15, 20138 Milan, Italy; IRCCS Multimedica, via G. Fantoli 16/15, 20138 Milan, Italy.
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14
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Karamali F, Esfahani MHN, Taleahmad S, Satarian L, Baharvand H. Stem cells from apical papilla promote differentiation of human pluripotent stem cells towards retinal cells. Differentiation 2018; 101:8-15. [DOI: 10.1016/j.diff.2018.02.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2017] [Revised: 02/14/2018] [Accepted: 02/23/2018] [Indexed: 12/14/2022]
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15
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Li R, Yu G, Azarin SM, Hubel A. Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells. Tissue Eng Part C Methods 2018; 24:289-299. [PMID: 29478388 DOI: 10.1089/ten.tec.2017.0531] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.
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Affiliation(s)
- Rui Li
- 1 Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota
| | - Guanglin Yu
- 2 Department of Mechanical Engineering, University of Minnesota , Minneapolis, Minnesota
| | - Samira M Azarin
- 3 Chemical Engineering and Materials Science, University of Minnesota , Minneapolis, Minnesota
| | - Allison Hubel
- 2 Department of Mechanical Engineering, University of Minnesota , Minneapolis, Minnesota
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16
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Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-free Condition. Methods Mol Biol 2018; 1502:143-58. [PMID: 26867543 DOI: 10.1007/7651_2015_318] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/22/2023]
Abstract
Recent advances in neural differentiation technology have paved the way to generate clinical grade neural progenitor populations from human pluripotent stem cells. These cells are an excellent source for the production of neural cell-based therapeutic products to treat incurable central nervous system disorders such as Parkinson's disease and spinal cord injuries. This progress can be complemented by the development of robust bioprocessing technologies for large scale expansion of clinical grade neural progenitors under GMP conditions for promising clinical use and drug discovery applications. Here, we describe a protocol for a robust, scalable expansion of human neural progenitor cells from pluripotent stem cells as 3D aggregates in a stirred suspension bioreactor. The use of this platform has resulted in easily expansion of neural progenitor cells for several passages with a fold increase of up to 4.2 over a period of 5 days compared to a maximum 1.5-2-fold increase in the adherent static culture over a 1 week period. In the bioreactor culture, these cells maintained self-renewal, karyotype stability, and cloning efficiency capabilities. This approach can be also used for human neural progenitor cells derived from other sources such as the human fetal brain.
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17
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Vernardis SI, Terzoudis K, Panoskaltsis N, Mantalaris A. Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor. Sci Rep 2017; 7:42138. [PMID: 28165055 PMCID: PMC5292706 DOI: 10.1038/srep42138] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Accepted: 01/03/2017] [Indexed: 12/19/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography–mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.
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Affiliation(s)
- Spyros I Vernardis
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK
| | - Konstantinos Terzoudis
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK
| | - Nicki Panoskaltsis
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK.,Department of Haematology, Imperial College, London, UK
| | - Athanasios Mantalaris
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK
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18
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Archibald PRT, Chandra A, Thomas D, Chose O, Massouridès E, Laâbi Y, Williams DJ. Comparability of automated human induced pluripotent stem cell culture: a pilot study. Bioprocess Biosyst Eng 2016; 39:1847-1858. [PMID: 27503483 PMCID: PMC5050253 DOI: 10.1007/s00449-016-1659-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2016] [Accepted: 07/29/2016] [Indexed: 11/27/2022]
Abstract
Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems’ CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories.
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Affiliation(s)
- Peter R T Archibald
- Centre for Biological Engineering, Loughborough University, Loughborough, LE11 3TU, UK.,Cell and Gene Therapy Platform CMC, GlaxoSmithKline PLC, Stevenage, UK
| | - Amit Chandra
- Centre for Biological Engineering, Loughborough University, Loughborough, LE11 3TU, UK.
| | - Dave Thomas
- TAP Biosystems, Part of the Sartorius Stedim Biotech Group, Royston, UK
| | - Olivier Chose
- CECS/I-Stem, AFM Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 2 rue Henri Desbruères, 91100, Corbeil-Essonnes, France
| | - Emmanuelle Massouridès
- CECS/I-Stem, AFM Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 2 rue Henri Desbruères, 91100, Corbeil-Essonnes, France
| | - Yacine Laâbi
- CECS/I-Stem, AFM Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 2 rue Henri Desbruères, 91100, Corbeil-Essonnes, France
| | - David J Williams
- Centre for Biological Engineering, Loughborough University, Loughborough, LE11 3TU, UK
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19
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Zhang L, Xu Y, Xu J, Wei Y, Xu X. Protein kinase A inhibitor, H89, significantly enhances survival rate of dissociated human embryonic stem cells following cryopreservation. Cell Prolif 2016; 49:589-98. [PMID: 27484641 DOI: 10.1111/cpr.12278] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2016] [Accepted: 07/01/2016] [Indexed: 12/30/2022] Open
Abstract
OBJECTIVES Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. MATERIALS AND METHODS Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow-freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESCs following cryopreservation. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89. RESULTS H89 significantly improved survival level of dissociated hESCs after cryopreservation through ROCK inhibition. H89-treated cells still maintained their pluripotency and differentiation capacity. CONCLUSIONS This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future.
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Affiliation(s)
- Liang Zhang
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yanqing Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China
| | - Jiandong Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China
| | - Yuping Wei
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China
| | - Xia Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China.
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20
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Meng G, Poon A, Liu S, Rancourt DE. An Effective and Reliable Xeno-free Cryopreservation Protocol for Single Human Pluripotent Stem Cells. Methods Mol Biol 2016; 1516:47-56. [PMID: 27032942 DOI: 10.1007/7651_2016_322] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/05/2023]
Abstract
Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined, xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free, single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 μM Rho-associated kinase inhibitor Y-27632 into two types of xeno-free, defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (~90 %) and cell expansion (~70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells.
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Affiliation(s)
- Guoliang Meng
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
| | - Anna Poon
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
| | - Shiying Liu
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
| | - Derrick E Rancourt
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
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21
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Slow Cooling Cryopreservation Optimized to Human Pluripotent Stem Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 951:57-65. [DOI: 10.1007/978-3-319-45457-3_5] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/06/2022]
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22
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Xu Y, Zhang L, Xu J, Wei Y, Xu X. Sensitivity of human embryonic stem cells to different conditions during cryopreservation. Cryobiology 2015; 71:486-92. [PMID: 26548334 DOI: 10.1016/j.cryobiol.2015.10.151] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 10/29/2015] [Accepted: 10/30/2015] [Indexed: 11/18/2022]
Abstract
Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.
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Affiliation(s)
- Yanqing Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
| | - Liang Zhang
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
| | - Jiandong Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
| | - Yuping Wei
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
| | - Xia Xu
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
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23
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ROCK inhibitor Y27632 promotes proliferation and diminishes apoptosis of marmoset induced pluripotent stem cells by suppressing expression and activity of caspase 3. Theriogenology 2015; 85:302-14. [PMID: 26476594 DOI: 10.1016/j.theriogenology.2015.09.020] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Revised: 08/31/2015] [Accepted: 09/08/2015] [Indexed: 12/16/2022]
Abstract
Previous studies reported that Rho-associated kinase inhibitor Y27632 markedly diminishes human embryonic stem cell and induced pluripotent stem cell (iPSC) dissociation-induced apoptosis and increases cloning efficiency in a feeder-free culture system. However, the mechanisms by which Y27632 protects pluripotent stem cells from apoptosis remain unknown. In the present study, we tested the effects of Y27632 on single dissociated marmoset iPSCs in a feeder-free culture. The results showed that Y27632 promoted the number of cells proliferating after passage by single-cell dissociation in a dose-dependent manner. The Rho-associated kinase inhibitor Y27632 markedly increased the cloning efficiency of marmoset iPSCs without affecting their karyotype and the expression of pluripotency markers. Meanwhile, Y27632 markedly diminished apoptosis of the marmoset iPSCs under even more severe conditions by suppressing the expression and activity of caspase 3. Taken together, the present results suggest that this reagent is effective in improving the cultural system of primate iPSCs.
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Zandi M, Muzaffar M, Shah SM, Kumar Singh M, Palta P, Kumar Singla S, Manik R, Chauhan MS. Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System. CELL JOURNAL 2015. [PMID: 26199905 PMCID: PMC4503840 DOI: 10.22074/cellj.2016.3728] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
OBJECTIVE In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. MATERIALS AND METHODS In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. RESULTS The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. CONCLUSION We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells.
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Affiliation(s)
- Mohammad Zandi
- Department of Animal and Poultry Science and Fisheries, Agricultural Institute, Iranian Research Organisation for Science and Technology (IROST), Tehran, Iran
| | - Musharifa Muzaffar
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Syed Mohmad Shah
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Manoj Kumar Singh
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Prabhat Palta
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Suresh Kumar Singla
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Radheysham Manik
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
| | - Manmohan Singh Chauhan
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India
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Abstract
This unit describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. Cryopreservation in E8 medium eliminates serum and other animal products, and is suitable for dealing with the increasing demand for high-quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, the protocols in this unit allow for efficient cryopreservation in a more time-saving manner.
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Affiliation(s)
- Weiwei Liu
- Faculty of Health Sciences, University of Macau, Taipa, Macau, China
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Lee JE, Lee DR. Human embryonic stem cells: derivation, maintenance and cryopreservation. Int J Stem Cells 2014; 4:9-17. [PMID: 24298329 DOI: 10.15283/ijsc.2011.4.1.9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/01/2011] [Indexed: 12/29/2022] Open
Abstract
Human embryonic stem cells (hESCs) are the most powerful candidate for the treatment of incurable diseases through the replacement of damaged cells and/or tissues in patients, although there are some obstacles to overcome for the clinical application of hESCs such as the assurance of guided differentiation and control of the immune response following cell therapy or tissue grafting. To obtain genetically stable hESCs and use them clinically, it is important to develop appropriate culture conditions. Additionally, the establishment of a hESC bank with a large number of hESC lines will be required for their clinical application because each hESC line is directed to have a different differentiation ability and immune characteristics such as HLA type. In this review, we describe the derivation and culture conditions of hESCs based on recent advances. Then, we will introduce several cryopreservation methods for hESCs, which is important for the development of cell bank.
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Imaizumi K, Nishishita N, Muramatsu M, Yamamoto T, Takenaka C, Kawamata S, Kobayashi K, Nishikawa SI, Akuta T. A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol. PLoS One 2014; 9:e88696. [PMID: 24533137 PMCID: PMC3922972 DOI: 10.1371/journal.pone.0088696] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Accepted: 01/10/2014] [Indexed: 12/18/2022] Open
Abstract
Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional −80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.
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Affiliation(s)
- Keitaro Imaizumi
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Kobe office, RIKEN Cell Tech Co. Ltd., Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Naoki Nishishita
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Marie Muramatsu
- Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Takako Yamamoto
- Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Chiemi Takenaka
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Shin Kawamata
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- * E-mail: (SK); (TA)
| | - Kenichiro Kobayashi
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Okura, Setagaya-ku, Tokyo, Japan
| | - Shin-ichi Nishikawa
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
| | - Teruo Akuta
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- Kobe office, RIKEN Cell Tech Co. Ltd., Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan
- * E-mail: (SK); (TA)
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Cohen RI, Thompson ML, Schryver B, Ehrhardt RO. Standardized cryopreservation of pluripotent stem cells. ACTA ACUST UNITED AC 2014; 28:1C.14.1-1C.14.10. [PMID: 24510767 DOI: 10.1002/9780470151808.sc01c14s28] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled-rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled-rate freezers. The protocol also eliminates the requirement for isopropanol, avoiding the hazards, on-going cost, and inconsistencies associated with its use and disposal. It provides a clinically relevant, inexpensive, reliable, and user-friendly method that successfully prepares cells for long-term cold storage and ensures maximum levels of cell viability post thaw.
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Gharechahi J, Pakzad M, Mirshavaladi S, Sharifitabar M, Baharvand H, Salekdeh GH. The effect of Rho-associated kinase inhibition on the proteome pattern of dissociated human embryonic stem cells. MOLECULAR BIOSYSTEMS 2014; 10:640-52. [DOI: 10.1039/c3mb70255c] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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Pakzad M, Ashtiani MK, Mousavi-Gargari SL, Baharvand H. Development of a simple, repeatable, and cost-effective extracellular matrix for long-term xeno-free and feeder-free self-renewal of human pluripotent stem cells. Histochem Cell Biol 2013; 140:635-48. [PMID: 24065274 DOI: 10.1007/s00418-013-1144-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/14/2013] [Indexed: 12/18/2022]
Abstract
Given the potential importance of human pluripotent stem cells (hPSCs) in translational research and regenerative medicine, the aim of the present study was to develop a simple, safe, and cost-effective substrate to expand hPSCs. We report the development of an extracellular matrix (ECM), designated "RoGel," based on conditioned medium (CM) of human fibroblasts under serum- and xeno-free culture conditions. The long-term self-renewal of hPSCs on RoGel was also assessed. The results showed that self-renewal, pluripotency, plating efficiency, and cloning efficiency of hPSCs on this newly developed ECM were similar to those of Matrigel, the conventional mouse-cell line-derived ECM. The cells had the capability to passage mechanically on a cold surface, which resulted in their long-term maintenance with normal karyotype. We have demonstrated that CM-coated plates preserved for 1 year at room temperature maintained the capability of hPSC expansion. This ECM provides an attractive hPSC culture platform for both research and future therapeutic applications.
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Affiliation(s)
- Mohammad Pakzad
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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Xia G, Santostefano KE, Goodwin M, Liu J, Subramony SH, Swanson MS, Terada N, Ashizawa T. Generation of neural cells from DM1 induced pluripotent stem cells as cellular model for the study of central nervous system neuropathogenesis. Cell Reprogram 2013; 15:166-77. [PMID: 23550732 DOI: 10.1089/cell.2012.0086] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.
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Affiliation(s)
- Guangbin Xia
- Department of Neurology, University of Florida, College of Medicine, Gainesville, FL 32611, USA
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32
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Taei A, Hassani SN, Eftekhari-Yazdi P, Rezazadeh Valojerdi M, Nokhbatolfoghahai M, Masoudi NS, Pakzad M, Gourabi H, Baharvand H. Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase β and Rho-associated kinase signaling. Hum Reprod 2013; 28:2661-71. [PMID: 23925393 DOI: 10.1093/humrep/det309] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
STUDY QUESTION Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? SUMMARY ANSWER Inhibition of glycogen synthase kinase β (GSK3β) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. WHAT IS KNOWN ALREADY Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. STUDY DESIGN, SIZE, DURATION In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3β inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. MAIN RESULTS AND THE ROLE OF CHANCE We found that 3 μM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. LIMITATIONS, REASONS FOR CAUTION The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. WIDER IMPLICATIONS OF THE FINDINGS This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. STUDY FUNDING/COMPETING INTEREST(S) This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.
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Affiliation(s)
- Adeleh Taei
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran
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Chen AKL, Chen X, Lim YM, Reuveny S, Oh SKW. Inhibition of ROCK-myosin II signaling pathway enables culturing of human pluripotent stem cells on microcarriers without extracellular matrix coating. Tissue Eng Part C Methods 2013; 20:227-38. [PMID: 23777438 DOI: 10.1089/ten.tec.2013.0191] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×10⁶ cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×10⁶ cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.
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Affiliation(s)
- Allen Kuan-Liang Chen
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , Singapore, Singapore
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Fattahi F, Asgari S, Pournasr B, Seifinejad A, Totonchi M, Taei A, Aghdami N, Salekdeh GH, Baharvand H. Disease-corrected hepatocyte-like cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol 2013; 54:863-73. [PMID: 23247991 DOI: 10.1007/s12033-012-9635-3] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.
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Affiliation(s)
- Faranak Fattahi
- Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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Lin PY, Yang YC, Hung SH, Lee SY, Lee MS, Chu IM, Hwang SM. Cryopreservation of human embryonic stem cells by a programmed freezer with an oscillating magnetic field. Cryobiology 2013; 66:256-60. [DOI: 10.1016/j.cryobiol.2013.02.061] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Revised: 02/19/2013] [Accepted: 02/20/2013] [Indexed: 11/25/2022]
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Li Y, Ma T. Bioprocessing of cryopreservation for large-scale banking of human pluripotent stem cells. Biores Open Access 2013; 1:205-14. [PMID: 23515461 PMCID: PMC3559214 DOI: 10.1089/biores.2012.0224] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process.
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Affiliation(s)
- Yan Li
- Department of Chemical and Biomedical Engineering, Florida State University , Tallahassee, Florida
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37
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Welte Y, Davies C, Schäfer R, Regenbrecht CRA. Patient derived cell culture and isolation of CD133⁺ putative cancer stem cells from melanoma. J Vis Exp 2013:e50200. [PMID: 23525090 DOI: 10.3791/50200] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression. Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin. Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133(+) CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133(+) CSCs and CD133(-) bulk, which can be cultivated and functionally analyzed thereafter.
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Affiliation(s)
- Yvonne Welte
- Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin.
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Xia G, Santostefano K, Hamazaki T, Liu J, Subramony SH, Terada N, Ashizawa T. Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro. J Mol Neurosci 2012; 51:237-48. [PMID: 23224816 DOI: 10.1007/s12031-012-9930-2] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2012] [Accepted: 11/21/2012] [Indexed: 12/27/2022]
Abstract
Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hampers therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2-induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary cultures of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within the neural lineage, neurons had the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared with control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. These differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.
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Affiliation(s)
- Guangbin Xia
- Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, USA
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Kurosawa H. Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells. J Biosci Bioeng 2012; 114:577-81. [PMID: 22898436 DOI: 10.1016/j.jbiosc.2012.07.013] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2012] [Revised: 06/12/2012] [Accepted: 07/23/2012] [Indexed: 11/19/2022]
Abstract
Susceptibility of human pluripotent stem cells (hPSCs), such as human iPS and embryonic stem (ES) cells, to single-cell dissociation has been a large obstacle to develop the efficient manipulation techniques required for stem cell research. When hPSCs are completely dissociated into single cells, programmed cell death (apoptosis) is immediately induced. A specific inhibitor of Rho-associated protein kinase (ROCK inhibitor), Y-27632, is of particular interest as a useful reagent that allows hPSCs to escape the dissociation-induced apoptosis. ROCK inhibitor has been used in a variety of applications associated with cell dissociation in the process of stem cell research, such as passaging, expansion, cryopreservation, gene transfer, differentiation induction, and cell sorting, suggesting that it may be a crucial reagent for the handling of hPSCs. This article reviews the current applications of ROCK inhibitors to stem cell research from the viewpoint of quality control of hPSCs.
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Affiliation(s)
- Hiroshi Kurosawa
- Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
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40
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Zahabi A, Shahbazi E, Ahmadieh H, Hassani SN, Totonchi M, Taei A, Masoudi N, Ebrahimi M, Aghdami N, Seifinejad A, Mehrnejad F, Daftarian N, Salekdeh GH, Baharvand H. A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev 2012; 21:2262-2272. [PMID: 22145677 DOI: 10.1089/scd.2011.0599] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.
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Affiliation(s)
- Azadeh Zahabi
- Department of Stem Cells and Developmental Biology, The Academic Center for Education, Culture and Research-ACECR, Tehran, Iran
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41
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T'Joen V, Cornelissen R. Xeno-free plant-derived hydrolysate-based freezing of human embryonic stem cells. Stem Cells Dev 2012; 21:1716-25. [PMID: 21867427 DOI: 10.1089/scd.2011.0374] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering and cell therapy. The large-scale banking of hESCs for research and future clinical application requires economic, defined, and xeno-free cryopreservation protocols. In this study, the possibility to substitute knockout serum replacement (KO-SR) in the cryopreservation process with vegetal and synthetic hydrolysates was investigated. To our knowledge, the use of hydrolysates in hESC cryopreservation has not been yet explored. Initially, 3 different hydrolysates (Ultrapep Soy, Hypep 4601 and EX-CELL(®) CD Hydrolysate Fusion) were tested in the cryopreservation solution. A concentration of 8 mg/mL EX-CELL CD Hydrolysate Fusion in the cryopreservation solution leads to the highest recovery ratio; thus, this solution was selected for additional cryopreservation experiments. To ensure reproducibility of the results, 3 hESC lines (HS181, H9, and BG01) were used. The hESCs were collected prefreezing by application of collagenase IV and cell dissociation solution. Experiments showed that it was feasible to substitute the KO-SR in both the cryopreservation solution as the thawing solution. The obtained recovery ratios were comparable to those obtained with KO-SR (no statistical significant difference; Student's t-test, P<0.05). Further optimization protocols showed a doubling of the obtained recovery ratio after addition of Rock-inhibitor Y-27632 post-thawing. The expansion profile and pluripotency analysis revealed no changes in normal hESC behavior. We conclude that the application of vegetal or synthetic hydrolysates is suitable for xeno-free hESC cryopreservation.
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Affiliation(s)
- Veronique T'Joen
- Department of Basic Medical Science, Faculty of Medicine and Health Science, Ghent University-UGent, Gent, Belgium.
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Abbasalizadeh S, Larijani MR, Samadian A, Baharvand H. Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor. Tissue Eng Part C Methods 2012; 18:831-51. [PMID: 22559864 DOI: 10.1089/ten.tec.2012.0161] [Citation(s) in RCA: 99] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (∼2×10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells" to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess parameters that are complemented by a simple and rapid scale-up platform.
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Affiliation(s)
- Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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43
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Niapour A, Karamali F, Nemati S, Taghipour Z, Mardani M, Nasr-Esfahani MH, Baharvand H. Cotransplantation of Human Embryonic Stem Cell-Derived Neural Progenitors and Schwann Cells in a Rat Spinal Cord Contusion Injury Model Elicits a Distinct Neurogenesis and Functional Recovery. Cell Transplant 2012; 21:827-843. [DOI: 10.3727/096368911x593163] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Cotransplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when cocultured with SCs in vitro and cotransplanted in a rat acute model of contused SCI. Cocultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs + SCs) relative to the vehicle and control groups. We also observed that animals receiving cotransplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation 5 weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the cotransplanted group when compared to the individual hESC-NP-grafted group. These findings have demonstrated that the cotransplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery.
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Affiliation(s)
- Ali Niapour
- Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
- Department of Anatomical Sciences, Ardebil University of Medical Science, Ardebil, Iran
| | - Fereshteh Karamali
- Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
| | - Shiva Nemati
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Zahra Taghipour
- Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Mohammad Mardani
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Mohammad Hossein Nasr-Esfahani
- Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
| | - 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, ACECR, Tehran, Iran
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DNA repair in human pluripotent stem cells is distinct from that in non-pluripotent human cells. PLoS One 2012; 7:e30541. [PMID: 22412831 PMCID: PMC3295811 DOI: 10.1371/journal.pone.0030541] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2011] [Accepted: 12/19/2011] [Indexed: 11/19/2022] Open
Abstract
The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use.
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45
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Chen VC, Couture SM, Ye J, Lin Z, Hua G, Huang HIP, Wu J, Hsu D, Carpenter MK, Couture LA. Scalable GMP compliant suspension culture system for human ES cells. Stem Cell Res 2012; 8:388-402. [PMID: 22459095 DOI: 10.1016/j.scr.2012.02.001] [Citation(s) in RCA: 101] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2011] [Revised: 02/06/2012] [Accepted: 02/10/2012] [Indexed: 02/06/2023] Open
Abstract
Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.
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Affiliation(s)
- Vincent C Chen
- Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.
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46
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Amirpour N, Karamali F, Rabiee F, Rezaei L, Esfandiari E, Razavi S, Dehghani A, Razmju H, Nasr-Esfahani MH, Baharvand H. Differentiation of Human Embryonic Stem Cell–Derived Retinal Progenitors into Retinal Cells by Sonic Hedgehog and/or Retinal Pigmented Epithelium and Transplantation into the Subretinal Space of Sodium Iodate–Injected Rabbits. Stem Cells Dev 2012; 21:42-53. [DOI: 10.1089/scd.2011.0073] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Noushin Amirpour
- Department of Cell and Molecular Biology, Royan Institute for Animal Biotechnology, Isfahan, Iran
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Fereshteh Karamali
- Department of Cell and Molecular Biology, Royan Institute for Animal Biotechnology, Isfahan, Iran
| | - Farzaneh Rabiee
- Department of Cell and Molecular Biology, Royan Institute for Animal Biotechnology, Isfahan, Iran
| | - Leila Rezaei
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Ebrahim Esfandiari
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Shahnaz Razavi
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Alireza Dehghani
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Hassan Razmju
- Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | | | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
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47
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Shahbazi E, Kiani S, Gourabi H, Baharvand H. Electrospun Nanofibrillar Surfaces Promote Neuronal Differentiation and Function from Human Embryonic Stem Cells. Tissue Eng Part A 2011; 17:3021-3031. [DOI: 10.1089/ten.tea.2011.0121] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Ebrahim Shahbazi
- Department of Stem Cells and Development al 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, ACECR, Tehran, Iran
| | - Sahar Kiani
- Department of Stem Cells and Development al Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hamid Gourabi
- Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Development al 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, ACECR, Tehran, Iran
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48
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Larijani MR, Seifinejad A, Pournasr B, Hajihoseini V, Hassani SN, Totonchi M, Yousefi M, Shamsi F, Salekdeh GH, Baharvand H. Retracted: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension. Stem Cells Dev 2011; 20:1911-1923. [DOI: 10.1089/scd.2010.0517] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Affiliation(s)
- Mehran Rezaei Larijani
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
| | - Ali Seifinejad
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
| | - Behshad Pournasr
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
| | - Vahid Hajihoseini
- Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
| | - Seydeh-Nafiseh Hassani
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
| | - Mehdi Totonchi
- Department of Genetics, Royan Institute for Reproductive Biomedicine, The Academic Center for Education, Culture and Research, Tehran, Iran
| | - Maryam Yousefi
- Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
| | - Farnaz Shamsi
- Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
| | - Ghasem Hosseini Salekdeh
- Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
- Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, The Academic Center for Education, Culture and Research, Tehran, Iran
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49
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Barbaric I, Jones M, Buchner K, Baker D, Andrews PW, Moore HD. Pinacidil enhances survival of cryopreserved human embryonic stem cells. Cryobiology 2011; 63:298-305. [PMID: 22027383 DOI: 10.1016/j.cryobiol.2011.10.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2011] [Revised: 09/22/2011] [Accepted: 10/06/2011] [Indexed: 12/20/2022]
Abstract
Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.
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Affiliation(s)
- Ivana Barbaric
- Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield, UK.
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50
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Ross AL, Leder DE, Weiss J, Izakovic J, Grichnik JM. Genomic instability in cultured stem cells: associated risks and underlying mechanisms. Regen Med 2011; 6:653-62. [DOI: 10.2217/rme.11.44] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Embryonic stem cells, mesenchymal stem cells and induced pluripotent stem cells expanded in vitro exhibit genomic instability. Commonly reported abnormalities include aneuploidy, deletions and duplications (including regions also amplified in cancer). Genomic instability confers an increased risk of malignant transformation that may impact the safety of cultured stem cell transplantation. Possible mechanisms responsible for this genomic instability include DNA repair mechanism abnormalities, telomere crisis, mitotic spindle abnormalities and inappropriate induction of meiotic pathways. Prior to widespread use of these cells in regenerative medicine, it will be critical to gain an understanding of the mechanisms responsible for genomic instability to develop strategies to prevent the accrual of chromosomal defects during expansion in vitro.
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Affiliation(s)
- Andrew L Ross
- Interdisciplinary Stem Cell Institute, University of Miami, Miller School of Medicine, Miami FL 33136, USA; Department of Dermatology, Melanoma Program Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Room 912, BRB, 1501 NW 10th Ave, Miami, FL 33136, USA
- Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Daniel E Leder
- Interdisciplinary Stem Cell Institute, University of Miami, Miller School of Medicine, Miami FL 33136, USA; Department of Dermatology, Melanoma Program Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Room 912, BRB, 1501 NW 10th Ave, Miami, FL 33136, USA
- Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Jonathan Weiss
- Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Jan Izakovic
- Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - James M Grichnik
- Interdisciplinary Stem Cell Institute, University of Miami, Miller School of Medicine, Miami FL 33136, USA; Department of Dermatology, Melanoma Program Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Room 912, BRB, 1501 NW 10th Ave, Miami, FL 33136, USA
- Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
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