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Azhdari M, Hausen AZ, Aghdami N, Baghaban-Eslaminejad M. Efficient and Reproducible Differentiation Protocol for Pluripotent Stem Cells into Functional Endothelial Cells: Unveiling the Path to Vascular Regeneration. Arch Med Res 2025; 56:103142. [PMID: 39837100 DOI: 10.1016/j.arcmed.2024.103142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2024] [Revised: 11/01/2024] [Accepted: 11/21/2024] [Indexed: 01/23/2025]
Abstract
INTRODUCTION Endothelial cells (ECs) play a crucial role in many treatments for cardiovascular diseases, such as blood vessel repair, tissue engineering, and drug delivery. The process of differentiating these cells is complex and involves various sources and numerous molecular and cellular events. Differentiating pluripotent stem cells (PSCs) into endothelial cells are one of the most effective sources for creating ECs in the lab and offers great potential for regenerative medicine. However, different cell types can appear during differentiation process. OBJECTIVES This study presents a reliable and reproducible protocol for efficiently differentiating human pluripotent stem cells (hPSCs) into mature endothelial cells with high purity (>98%). METHODS FLK1+ cells were isolated from hPSCs using fluorescence-activated cell sorting (FACS). Then isolated FLK1+ cells differentiated into high-purity endothelial cells (ECs) by adding endothelial growth factors (VEGF, FGF, and EGM-2 medium). The differentiated ECs were extensively characterized by evaluating key endothelial markers and assessing their functional abilities, such as tube formation and response to angiogenic signals. Finally, the ECs were further purified using a second FACS step with a CD31 antibody. RESULTS The differentiated hPSC-derived endothelial cells (hPSC-ECs) expressed high levels of PECAM-1 (CD31), VE-cadherin (CD144), and von Willebrand factor (vWF), with more than 98% of the cells showing these markers. Additionally, the hPSC-ECs formed tubular structures and effectively took up acetylated fluorescently-labeled low-density lipoprotein (DiI-ac-LDL), demonstrating their functionality as endothelial cells. CONCLUSION Our study clarifies the molecular mechanisms involved in the differentiation of hPSCs into endothelial cells, emphasizing key signaling pathways important for determining endothelial cell fate. These findings provide a framework for the scalable production of transplantable endothelial cells, representing a significant advancement in personalized therapies and tissue engineering for regenerative medicine.
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Affiliation(s)
- Manizheh Azhdari
- Department of Pathology, School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research, Tehran, Iran.
| | - Axel Zur Hausen
- Department of Pathology, School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran; Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran
| | - Mohamadreza Baghaban-Eslaminejad
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran; Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran
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Kimura K, Tsukamoto M, Sugisaki H, Tanaka M, Kuwamura M, Matsumoto Y, Ishihara G, Watanabe K, Okada M, Nakanishi M, Sugiura K, Hatoya S. Generation of footprint-free, high-quality feline induced pluripotent stem cells using Sendai virus vector. Regen Ther 2024; 26:708-716. [PMID: 39286639 PMCID: PMC11403254 DOI: 10.1016/j.reth.2024.08.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2024] [Revised: 08/04/2024] [Accepted: 08/18/2024] [Indexed: 09/19/2024] Open
Abstract
Companion animals, such as felines and canines, could provide an excellent platform for translational research from veterinary to human medicine. However, the use of feline induced pluripotent stems (fiPSCs) of quality in basic or clinical research has not been reported. Here, we generated footprint-free fiPSCs derived from embryonic cells, as well as juvenile feline uterus-derived cells using Sendai virus vector harboring six feline-specific pluripotency-associated genes. The fiPSCs were confirmed to be of high quality with the potential to form teratomas including all three germ layers. Furthermore, our fiPSCs were maintained under feeder-free and chemically-defined conditions using StemFit® AK02N and recombinant laminin 511, iMatrix-511. Further research on fiPSCs could result in their widespread application in veterinary regenerative medicine, which could pave the way for their use in advanced regenerative medicine research for humans.
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Affiliation(s)
- Kazuto Kimura
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Masaya Tsukamoto
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Hiroko Sugisaki
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Miyuu Tanaka
- Department of Integrated Structural Biosciences, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Mitsuru Kuwamura
- Department of Integrated Structural Biosciences, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Yuki Matsumoto
- Anicom Specialty Medical Institute, Inc., Shinjuku-ku, Tokyo 231-0033, Japan
| | - Genki Ishihara
- Anicom Specialty Medical Institute, Inc., Shinjuku-ku, Tokyo 231-0033, Japan
| | - Kei Watanabe
- Anicom Specialty Medical Institute, Inc., Shinjuku-ku, Tokyo 231-0033, Japan
| | - Mika Okada
- TOKIWA-Bio, Inc., Tsukuba, Ibaraki 305-0047, Japan
| | | | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
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KIMURA K, NAGAKURA H, TSUKAMOTO M, YOSHIDA T, SUGISAKI H, SHISHIDA K, TACHI Y, SHIMASAKI S, SUGIURA K, HATOYA S. Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems. J Vet Med Sci 2024; 86:247-257. [PMID: 38171744 PMCID: PMC10963097 DOI: 10.1292/jvms.23-0422] [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: 10/10/2023] [Accepted: 12/27/2023] [Indexed: 01/05/2024] Open
Abstract
Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.
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Affiliation(s)
- Kazuto KIMURA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Hiroya NAGAKURA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Masaya TSUKAMOTO
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Takumi YOSHIDA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Hiroko SUGISAKI
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Kohei SHISHIDA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Yuta TACHI
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Shoko SHIMASAKI
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Kikuya SUGIURA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
| | - Shingo HATOYA
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan
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Netsrithong R, Garcia-Perez L, Themeli M. Engineered T cells from induced pluripotent stem cells: from research towards clinical implementation. Front Immunol 2024; 14:1325209. [PMID: 38283344 PMCID: PMC10811463 DOI: 10.3389/fimmu.2023.1325209] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Accepted: 12/15/2023] [Indexed: 01/30/2024] Open
Abstract
Induced pluripotent stem cell (iPSC)-derived T (iT) cells represent a groundbreaking frontier in adoptive cell therapies with engineered T cells, poised to overcome pivotal limitations associated with conventional manufacturing methods. iPSCs offer an off-the-shelf source of therapeutic T cells with the potential for infinite expansion and straightforward genetic manipulation to ensure hypo-immunogenicity and introduce specific therapeutic functions, such as antigen specificity through a chimeric antigen receptor (CAR). Importantly, genetic engineering of iPSC offers the benefit of generating fully modified clonal lines that are amenable to rigorous safety assessments. Critical to harnessing the potential of iT cells is the development of a robust and clinically compatible production process. Current protocols for genetic engineering as well as differentiation protocols designed to mirror human hematopoiesis and T cell development, vary in efficiency and often contain non-compliant components, thereby rendering them unsuitable for clinical implementation. This comprehensive review centers on the remarkable progress made over the last decade in generating functional engineered T cells from iPSCs. Emphasis is placed on alignment with good manufacturing practice (GMP) standards, scalability, safety measures and quality controls, which constitute the fundamental prerequisites for clinical application. In conclusion, the focus on iPSC as a source promises standardized, scalable, clinically relevant, and potentially safer production of engineered T cells. This groundbreaking approach holds the potential to extend hope to a broader spectrum of patients and diseases, leading in a new era in adoptive T cell therapy.
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Affiliation(s)
- Ratchapong Netsrithong
- Department of Hematology, Amsterdam University Medical Center (UMC), Vrije Universiteit Amsterdam, Amsterdam, Netherlands
- Cancer Biology and Immunology, Cancer Center Amsterdam, Amsterdam, Netherlands
| | - Laura Garcia-Perez
- Department of Hematology, Amsterdam University Medical Center (UMC), Vrije Universiteit Amsterdam, Amsterdam, Netherlands
- Cancer Biology and Immunology, Cancer Center Amsterdam, Amsterdam, Netherlands
| | - Maria Themeli
- Department of Hematology, Amsterdam University Medical Center (UMC), Vrije Universiteit Amsterdam, Amsterdam, Netherlands
- Cancer Biology and Immunology, Cancer Center Amsterdam, Amsterdam, Netherlands
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Ali HRW, Suliman S, Osman TAH, Carrasco M, Bruland O, Costea DE, Ræder H, Mustafa K. Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues. Stem Cell Res Ther 2023; 14:199. [PMID: 37559144 PMCID: PMC10410907 DOI: 10.1186/s13287-023-03403-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 06/15/2023] [Indexed: 08/11/2023] Open
Abstract
BACKGROUND Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness. METHODS iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods. RESULTS More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective "whitish" outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type. CONCLUSIONS The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.
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Affiliation(s)
- Hassan R W Ali
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Salwa Suliman
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Tarig Al-Hadi Osman
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway
| | - Manuel Carrasco
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
- Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway
| | - Ove Bruland
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Daniela-Elena Costea
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
- Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway
- Gade Laboratory for Pathology, Haukeland University Hospital, Bergen, Norway
| | - Helge Ræder
- Department of Clinical Science, University of Bergen, Bergen, Norway.
- Department of Pediatrics, Haukeland University Hospital, Bergen, Norway.
| | - Kamal Mustafa
- Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway.
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Li J, Wu Y, Yao X, Tian Y, Sun X, Liu Z, Ye X, Wu C. Preclinical Research of Stem Cells: Challenges and Progress. Stem Cell Rev Rep 2023:10.1007/s12015-023-10528-y. [PMID: 37097496 DOI: 10.1007/s12015-023-10528-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/06/2023] [Indexed: 04/26/2023]
Abstract
In recent years, great breakthroughs have been made in basic research and clinical applications of stem cells in regenerative medicine and other fields, which continue to inspire people to explore the field of stem cells. With nearly unlimited self-renewal ability, stem cells can generate at least one type of highly differentiated daughter cell, which provides broad development prospects for the treatment of human organ damage and other diseases. In the field of stem cell research, related technologies for inducing or isolating stem cells are relatively mature, and a variety of stable stem cell lines have been successfully constructed. To realize the full clinical application of stem cells as soon as possible, it is more and more important to further optimize each stage of stem cell research while conforming to Current Good Manufacture Practices (cGMP) standards. Here, we synthesized recent developments in stem cell research and focus on the introduction of xenogenicity in the preclinical research process and the remaining problems of various cell bioreactors. Our goal is to promote the development of technologies for xeno-free culture and clinical expansion of stem cells through in-depth discussion of current research. This review will provide new insight into stem cell research protocols and will contribute to the creation of efficient and stable stem cell expansion systems.
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Affiliation(s)
- Jinhu Li
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yurou Wu
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xiang Yao
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yao Tian
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xue Sun
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Zibo Liu
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xun Ye
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Chunjie Wu
- Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
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Schjeide BMM, Püschel GP. Determining On-Target, Off-Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe-Harbor Modification of iPSCs Using Double-Control Quantitative Copy Number PCR. Curr Protoc 2023; 3:e635. [PMID: 36598341 DOI: 10.1002/cpz1.635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.
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Affiliation(s)
- Brit-Maren Michaud Schjeide
- Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany.,Department of Nutritional Biochemistry, Faculty of Life Sciences: Food, Nutrition and Health, University of Bayreuth, Kulmbach, Germany
| | - Gerhard Paul Püschel
- Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany
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Ghorbani-Dalini S, Azarpira N, Sangtarash MH, Urbach V, Yaghobi R, Soleimanpour-Lichaei HR, Sarshar M. Optimization of 3D islet-like cluster derived from human pluripotent stem cells: an efficient in vitro differentiation protocol. Gene 2022; 845:146855. [PMID: 36058497 DOI: 10.1016/j.gene.2022.146855] [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: 06/11/2022] [Revised: 08/24/2022] [Accepted: 08/26/2022] [Indexed: 11/18/2022]
Abstract
Development of an optimized protocol to produce sufficient functional human insulin-producing islet-like cluster is important as a potential therapeutic strategy for diabetes as well as in vitro studies. Here, we described a stepwise protocol for differentiation of the human induced pluripotent stem cell line (R1-hiPSC1) into the islet-like cluster using several growth factors and small molecules. Therefore, various differentiation steps have been adopted to maximize mimicking of developmental processes in order to form functional islet like cluster. The differentiation protocol enables us to generate 3D islet-like clusters with highly viable cells, which are insulin producer and glucose responsive. Transcriptome analysis of transcription factors and functional genes revealed high coordination between gene expressions and resembling to those reported during natural development of islet cell. This coordination was further confirmed by hierarchical clustering of genes during differentiation. Furthermore, the islet-like clusters were enriched with insulin producing cells and formed glucose responsiveness behavior upon stimulation with glucose. Our protocol provides a robust platform and well-behaved model for additional developmental studies and shed light our clusters as a good candidate for in vitro model. Further studies are needed to assess the hormonal content of this cluster as well as transplantation into the animal model.
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Affiliation(s)
- Sadegh Ghorbani-Dalini
- Department of Research and Development, CBSAlife Ltd., Richardson Center of Food Technology and Research, Winnipeg, Manitoba, Canada; Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Negar Azarpira
- Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
| | | | - Valérie Urbach
- Insitut National de la Santé Et de la Recherche Médicale, U1151 Paris, France
| | - Ramin Yaghobi
- Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hamid Reza Soleimanpour-Lichaei
- Department of Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
| | - Meysam Sarshar
- Research Laboratories, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy
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Shayestehfar M, Farahi S, Kheiri Yeganeh Azar B, Memari A, Baluchnejadmojarad T, Faghihi F. Generating Human Induced Pluripotent Stem Cell Via Low-Dose Polyethylenimine-Mediated Transfection: An Optimized Protocol. DNA Cell Biol 2022; 41:903-916. [PMID: 35984994 DOI: 10.1089/dna.2022.0331] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Human dermal fibroblasts (HDFs) can be reprogrammed through different strategies to generate human induced pluripotent stem cells (hiPSCs). However, most of these strategies require high-cost materials and specific equipment not readily accessible in most laboratories. Hence, liposomal and virus-based techniques can replace with polyethylenimine (PEI)-mediated transfection to overcome these challenges. However, few researchers have addressed the PEI's ability to transfect HDFs. This study used PEI reagent to transfer oriP/EBNA1-based vector into HDFs to produce hiPSC lines. We first described conditions allowing the efficient transfection of HDFs with low cytotoxicity and without specific types of equipment and optimized several parameters relevant to the transfection procedure. We then monitored the effect of different N/P ratios on transfection efficiency and cytotoxicity using flow cytometry and fluorescent microscopy. By the results, we found that transfection efficiency was greatly affected by plasmid DNA concentration, PEI concentration, order of combining reagents, serum presence in polyplexes, and the duration of serum starvations. Moreover, using the optimized condition, we found that the N/P ratio of 3 achieved the highest percentage of HDFs positive for green fluorescent protein plasmid (∼40%) with minimal cell toxicity. We finally generated hiPSCs using the optimized protocol and oriP/EBNA1-based vectors. We confirmed hiPSC formation by characterizing tests: alkaline phosphatase staining, immunocytochemistry assay, real-time PCR analysis, in vitro differentiation into three germ layers, and karyotyping test. In conclusion, our results indicated that 25 kDa branched PEI could efficiently transfect HDFs toward generating hiPSCs via a simple, cost-effective, and optimized condition.
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Affiliation(s)
- Monir Shayestehfar
- Department of Neuroscience, Faculty of Advanced Technology in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Sara Farahi
- Biotechnology Department, Shahid Beheshti University of medical science, Tehran, Iran
| | - Behjat Kheiri Yeganeh Azar
- Department of Molecular Medicine, Faculty of Advanced Technology in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Amirhossein Memari
- Sports Medicine Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
| | | | - Faezeh Faghihi
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
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Hua Y, Yoshimochi K, Li J, Takekita K, Shimotsuma M, Li L, Qu X, Zhang J, Sawa Y, Liu L, Miyagawa S. Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells. Stem Cell Res Ther 2022; 13:223. [PMID: 35658933 PMCID: PMC9166585 DOI: 10.1186/s13287-022-02879-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2022] [Accepted: 05/03/2022] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost. METHODS In this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down's syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2. RESULTS We found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers. CONCLUSIONS Our xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.
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Affiliation(s)
- Ying Hua
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Kenji Yoshimochi
- NACALAI TESQUE, INC. Research and Development Department, Kyoto, 604-0855, Japan
| | - Junjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan.,Division of Cardiovascular Surgery, Department of Design for Tissue Regeneration, Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Kazuhiro Takekita
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Motoshi Shimotsuma
- NACALAI TESQUE, INC. Research and Development Department, Kyoto, 604-0855, Japan
| | - Lingjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Xiang Qu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Jingbo Zhang
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | | | - Li Liu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan. .,Division of Cardiovascular Surgery, Department of Design for Tissue Regeneration, Graduate School of Medicine, Osaka, 565-0871, Japan.
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan.
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11
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Rossbach B, Hariharan K, Mah N, Oh SJ, Volk HD, Reinke P, Kurtz A. Human iPSC-Derived Renal Cells Change Their Immunogenic Properties during Maturation: Implications for Regenerative Therapies. Cells 2022; 11:cells11081328. [PMID: 35456007 PMCID: PMC9032821 DOI: 10.3390/cells11081328] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/11/2022] [Accepted: 04/12/2022] [Indexed: 12/24/2022] Open
Abstract
The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.
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Affiliation(s)
- Bella Rossbach
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
| | - Krithika Hariharan
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer Project Center for Stem Cell Processing, 97082 Würzburg, Germany
| | - Nancy Mah
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
| | - Su-Jun Oh
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
| | - Hans-Dieter Volk
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Institute for Medical Immunology (IMI), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Petra Reinke
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Berlin Center for Advanced Therapies (BeCat), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Andreas Kurtz
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
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12
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Flechner M, Schaller J, Stahl M, Achberger K, Gerike S, Hannappel Y, Fu J, Jaeger M, Hellweg T, Duschl C, Uhlig K. Adhesion, proliferation and detachment of various cell types on thermoresponsive microgel coatings. Biotechnol Bioeng 2022; 119:1728-1739. [PMID: 35355251 DOI: 10.1002/bit.28095] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/23/2022] [Accepted: 03/24/2022] [Indexed: 11/07/2022]
Abstract
Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for non-specific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 3 7 ∘ C, but become repulsive upon a cell-compatible temperature drop to e.g. 3 2 ∘ C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells (iPS-cells) and cells in serum-free culture on. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for e.g. stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Marie Flechner
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany
| | - Julia Schaller
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany
| | - Maike Stahl
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany
| | - Kevin Achberger
- Eberhard Karls University Tuebingen, Institute of Neuroanatomy & Developmental Biology INDB, 72074, Tuebingen, Germany
| | - Susanna Gerike
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany
| | - Yvonne Hannappel
- Department of Physical and Biophysical Chemistry (PC III), Bielefeld University, 33615, Bielefeld, Germany
| | - Jianan Fu
- PAN-Biotech GmbH, 94501, Aidenbach, Germany
| | - Magnus Jaeger
- German Federal Institute for Risk Assessment (BfR), 10589, Berlin, Germany
| | - Thomas Hellweg
- Department of Physical and Biophysical Chemistry (PC III), Bielefeld University, 33615, Bielefeld, Germany
| | - Claus Duschl
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany
| | - Katja Uhlig
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany.,German Federal Institute for Risk Assessment (BfR), 10589, Berlin, Germany
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13
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Aghasizadeh M, Moghaddam T, Bahrami AR, Sadeghian H, Alavi SJ, Matin MM. 8-Geranyloxycarbostyril as a potent 15-LOX-1 inhibitor showed great anti-tumor effects against prostate cancer. Life Sci 2022; 293:120272. [PMID: 35065164 DOI: 10.1016/j.lfs.2021.120272] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 12/20/2021] [Accepted: 12/23/2021] [Indexed: 10/19/2022]
Abstract
Carbostyrils are quinolone derivatives, with possible growth inhibition properties on cancer cells. Unlike many tumors, 15-Lipoxygenase-1 (15-LOX-1) is highly expressed in prostate cancer (PCa) cells and has oncogenic properties. Here, with the hypothesis that 6-, 7- and 8-geranyloxycarbostyril (GQ) have inhibitory properties on 15-LOX-1, their effects were assessed on PCa cells. Their cytotoxic effects were evaluated by MTT assay and mechanism of cell death was investigated using annexin V/PI staining. Finally, the anti-tumor properties of 8-GQ were assessed in immunocompromised C57BL/6 mice bearing human PCa cells. Accordingly, these compounds could effectively inhibit 15-LOX activity in PCa cells. MTT and flow cytometry tests confirmed their toxic effects on PCa cells, with no significant toxicity on normal cells, and apoptosis was the main mechanism of cell death. In vivo results indicated that use of 8-GQ at 50 mg/kg had stronger anti-tumor effects than 5 mg/kg cisplatin, with fewer side effects on normal tissues. Therefore, 8-GQ can be introduced as a potential drug candidate with 15-LOX-1 inhibitory potency, which can be effective in treatment of prostate cancer, and should be considered for further drug screening investigations.
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Affiliation(s)
- Mehrdad Aghasizadeh
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Tayebe Moghaddam
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Ahmad Reza Bahrami
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Hamid Sadeghian
- Neurogenic Inflammation Research Center, Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Seyed Jamal Alavi
- Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Maryam M Matin
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Novel Diagnostics and Therapeutics Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran; Stem Cell and Regenerative Medicine Research Group, Iranian Academic Center for Education, Culture and Research (ACECR), Khorasan Razavi Branch, Mashhad, Iran.
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14
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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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15
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Ahani-Nahayati M, Niazi V, Moradi A, Pourjabbar B, Roozafzoon R, Baradaran-Rafii A, Keshel SH. Cell-based therapy for ocular disorders: A promising frontier. Curr Stem Cell Res Ther 2021; 17:147-165. [PMID: 34161213 DOI: 10.2174/1574888x16666210622124555] [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: 12/17/2020] [Revised: 02/06/2021] [Accepted: 04/19/2021] [Indexed: 11/22/2022]
Abstract
As the ocular disorders causing long-term blindness or optical abnormalities of the ocular tissue affect the quality of life of patients to a large extent, awareness of their corresponding pathogenesis and the earlier detection and treatment need more consideration. Though current therapeutics result in desirable outcomes, they do not offer an inclusive solution for development of visual impairment to blindness. Accordingly, stem cells, because of their particular competencies, have gained extensive attention for application in regenerative medicine of ocular diseases. In the last decades, a wide spectrum of stem cells surrounding mesenchymal stem/stromal cells (MSC), neural stem cells (NSCs), and embryonic/induced pluripotent stem cells (ESCs/iPSCs) accompanied by Müller glia, ciliary epithelia-derived stem cells, and retinal pigment epithelial (RPE) stem cells have been widely investigated to report their safety and efficacy in preclinical models and also human subjects. In this regard, in the first interventions, RPE cell suspensions were successfully utilized to ameliorate visual defects of the patients suffering from age-related macular degeneration (AMD) after subretinal transplantation. Herein, we will explain the pathogenesis of ocular diseases and highlight the novel discoveries and recent findings in the context of stem cell-based therapies in these disorders, focusing on the in vivo reports published during the last decade.
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Affiliation(s)
- Milad Ahani-Nahayati
- Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
| | - Vahid Niazi
- Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
| | - Alireza Moradi
- Department of Physiology, School of Medicine, Iran University of Medical Science, Tehran, Iran
| | - Bahareh Pourjabbar
- Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
| | - Reza Roozafzoon
- Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
| | | | - Saeed Heidari Keshel
- Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
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16
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Kimura K, Tsukamoto M, Yoshida T, Tanaka M, Kuwamura M, Ohtaka M, Nishimura K, Nakanishi M, Sugiura K, Hatoya S. Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions. Mol Reprod Dev 2021; 88:395-404. [PMID: 34010985 DOI: 10.1002/mrd.23478] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Revised: 04/11/2021] [Accepted: 05/05/2021] [Indexed: 12/12/2022]
Abstract
Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.
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Affiliation(s)
- Kazuto Kimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Masaya Tsukamoto
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Takumi Yoshida
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Miyuu Tanaka
- Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Mitsuru Kuwamura
- Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | | | - Ken Nishimura
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Mahito Nakanishi
- TOKIWA-Bio, Inc., Tsukuba, Japan.,National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
| | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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17
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Carelli S, Giallongo T, Rey F, Barzaghini B, Zandrini T, Pulcinelli A, Nardomarino R, Cerullo G, Osellame R, Cereda C, Zuccotti GV, Raimondi MT. Neural precursors cells expanded in a 3D micro-engineered niche present enhanced therapeutic efficacy in vivo. Nanotheranostics 2021; 5:8-26. [PMID: 33391972 PMCID: PMC7738947 DOI: 10.7150/ntno.50633] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Accepted: 09/04/2020] [Indexed: 12/13/2022] Open
Abstract
Rationale: Stem Cells (SCs) show a great potential in therapeutics for restoring and regenerating native tissues. The clinical translation of SCs therapies is currently hindered by the inability to expand SCs in vitro in large therapeutic dosages, while maintaining their safety and potency. The use of biomaterials allows for the generation of active biophysical signals for directing SCs fate through 3D micro-scaffolds, such as the one named “Nichoid”, fabricated with two-photon laser polymerization with a spatial resolution of 100 nm. The aims of this study were: i) to investigate the proliferation, differentiation and stemness properties of neural precursor cells (NPCs) following their cultivation inside the Nichoid micro-scaffold; ii) to assess the therapeutic effect and safety in vivo of NPCs cultivated in the Nichoid in a preclinical experimental model of Parkinson's Disease (PD). Methods: Nichoids were fabricated by two photon laser polymerization onto circular glass coverslips using a home-made SZ2080 photoresist. NPCs were grown inside the Nichoid for 7 days, counted and characterized with RNA-Seq, Real Time PCR analysis, immunofluorescence and Western Blot. Then, NPCs were transplanted in a murine experimental model of PD, in which parkinsonism was induced by the intraperitoneal administration of the neurotoxin MPTP in C57/bl mice. The efficacy of engrafted Nichoid-expanded NPCs was evaluated by means of specific behavioral tests and, after animal sacrifice, with immunohistochemical studies in brain slices. Results: NPCs grown inside the Nichoid show a significantly higher cell viability and proliferation than in standard culture conditions in suspension. Furthermore, we report the mechanical conditioning of NPCs in 3D micro-scaffolds, showing a significant increase in the expression of pluripotency genes. We also report that such mechanical reprogramming of NPCs produces an enhanced therapeutic effect in the in vivo model of PD. Recovery of PD symptoms was significantly increased when animals were treated with Nichoid-grown NPCs, and this is accompanied by the recovery of dopaminergic markers expression in the striatum of PD affected mice. Conclusion: SCs demonstrated an increase in pluripotency potential when expanded inside the Nichoid, without the need of any genetic modification of cells, showing great promise for large-scale production of safe and functional cell therapies to be used in multiple clinical applications.
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Affiliation(s)
- Stephana Carelli
- Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", L. Sacco Department of Biomedical and Clinical Sciences, University of Milano, Milano, 20157, Italy
| | - Toniella Giallongo
- Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", L. Sacco Department of Biomedical and Clinical Sciences, University of Milano, Milano, 20157, Italy
| | - Federica Rey
- Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", L. Sacco Department of Biomedical and Clinical Sciences, University of Milano, Milano, 20157, Italy
| | - Bianca Barzaghini
- Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, 20133, Italy
| | - Tommaso Zandrini
- Istituto di Fotonica e Nanotecnologie (IFN)-CNR and Department of Physics, Politecnico di Milano, Milano, 20133, Italy
| | - Andrea Pulcinelli
- Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, 20133, Italy
| | - Riccardo Nardomarino
- Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, 20133, Italy
| | - Giulio Cerullo
- Istituto di Fotonica e Nanotecnologie (IFN)-CNR and Department of Physics, Politecnico di Milano, Milano, 20133, Italy
| | - Roberto Osellame
- Istituto di Fotonica e Nanotecnologie (IFN)-CNR and Department of Physics, Politecnico di Milano, Milano, 20133, Italy
| | - Cristina Cereda
- Genomic and Postgenomic Lab, IRCCS Mondino Foundation, Pavia, 27100, Italy
| | - Gian Vincenzo Zuccotti
- Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", L. Sacco Department of Biomedical and Clinical Sciences, University of Milano, Milano, 20157, Italy
| | - Manuela Teresa Raimondi
- Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, 20133, Italy
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18
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Taei A, Kiani T, Taghizadeh Z, Moradi S, Samadian A, Mollamohammadi S, Sharifi‐Zarchi A, Guenther S, Akhlaghpour A, Asgari Abibeiglou B, Najar‐Asl M, Karamzadeh R, Khalooghi K, Braun T, Hassani S, Baharvand H. Temporal activation of LRH-1 and RAR-γ in human pluripotent stem cells induces a functional naïve-like state. EMBO Rep 2020; 21:e47533. [PMID: 33252195 PMCID: PMC7534641 DOI: 10.15252/embr.201847533] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Revised: 06/13/2020] [Accepted: 07/17/2020] [Indexed: 12/19/2022] Open
Abstract
Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-β signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-β signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.
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Affiliation(s)
- Adeleh Taei
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
- Department of Developmental BiologyUniversity of Science and CultureTehranIran
| | - Tahereh Kiani
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Zeinab Taghizadeh
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Sharif Moradi
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Azam Samadian
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Sepideh Mollamohammadi
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Ali Sharifi‐Zarchi
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
- Computer Engineering DepartmentSharif University of TechnologyTehranIran
| | - Stefan Guenther
- Department of Cardiac Development and RemodelingMax‐Planck Institute for Heart and Lung ResearchBad NauheimGermany
| | - Azimeh Akhlaghpour
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Behrouz Asgari Abibeiglou
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Mostafa Najar‐Asl
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Razieh Karamzadeh
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Keynoosh Khalooghi
- Department of Cardiac Development and RemodelingMax‐Planck Institute for Heart and Lung ResearchBad NauheimGermany
| | - Thomas Braun
- Department of Cardiac Development and RemodelingMax‐Planck Institute for Heart and Lung ResearchBad NauheimGermany
| | - Seyedeh‐Nafiseh Hassani
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and TechnologyACECRTehranIran
- Department of Developmental BiologyUniversity of Science and CultureTehranIran
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19
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Continuous ES/Feeder Cell-Sorting Device Using Dielectrophoresis and Controlled Fluid Flow. MICROMACHINES 2020; 11:mi11080734. [PMID: 32751153 PMCID: PMC7464685 DOI: 10.3390/mi11080734] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 07/16/2020] [Accepted: 07/26/2020] [Indexed: 12/21/2022]
Abstract
Pluripotent stem cells (PSCs) are considered as being an important cell source for regenerative medicine. The culture of PSCs usually requires a feeder cell layer or cell adhesive matrix coating such as Matrigel, laminin, and gelatin. Although a feeder-free culture using a matrix coating has been popular, the on-feeder culture is still an effective method for the fundamental study of regenerative medicine and stem cell biology. To culture PSCs on feeder cell layers, the elimination of feeder cells is required for biological or gene analysis and for cell passage. Therefore, a simple and cost-effective cell sorting technology is required. There are several commercialized cell-sorting methods, such as FACS or MACS. However, these methods require cell labeling by fluorescent dye or magnetic antibodies with complicated processes. To resolve these problems, we focused on dielectrophoresis (DEP) phenomena for cell separation because these do not require any fluorescent or magnetic dyes or antibodies. DEP imposes an electric force on living cells under a non-uniform AC electric field. The direction and magnitude of the DEP force depend on the electric property and size of the cell. Therefore, DEP is considered as a promising approach for sorting PSCs from feeder cells. In this study, we developed a simple continuous cell-sorting device using the DEP force and fluid-induced shear force. As a result, mouse embryonic stem cells (mESCs) were purified from a mixed-cell suspension containing mESCs and mouse embryonic fibroblasts (MEFs) using our DEP cell-sorting device.
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20
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Kasai K, Tohyama S, Suzuki H, Tanosaki S, Fukuda K, Fujita J, Miyata S. Cost-effective culture of human induced pluripotent stem cells using UV/ozone-modified culture plastics with reduction of cell-adhesive matrix coating. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 111:110788. [PMID: 32279811 DOI: 10.1016/j.msec.2020.110788] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2018] [Revised: 02/03/2020] [Accepted: 02/28/2020] [Indexed: 11/30/2022]
Abstract
Human induced pluripotent stem cells (hiPSCs) are considered to be one of the most promising cell resources for regenerative medicine. HiPSCs usually maintain their pluripotency when they are cultured on feeder cell layers or are attached to a cell-adhesive extracellular matrix. In this study, we developed a culture system based on UV/ozone modification for conventional cell culture plastics to generate a suitable surface condition for hiPSCs. Time of flight secondary ion mass spectrometry (ToF-SIMS) was carried out to elucidate the relationship between hiPSC adhesion and UV/ozone irradiation-induced changes to surface chemistry of cell culture plastics. Cell culture plastics with modified surfaces enabled growth of a feeder-free hiPSC culture with markedly reduced cell-adhesive matrix coating. Our cell culture system using UV/ozone-modified cell culture plastics may produce clinically relevant hiPSCs at low costs, and can be easily scaled up in culture systems to produce a large number of hiPSCs.
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Affiliation(s)
- Kohei Kasai
- Graduate School of Science and Technology, Keio University, 3-14-1 Kohoku-ku, Yokohama 223-8522, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hayato Suzuki
- Graduate School of Science and Technology, Keio University, 3-14-1 Kohoku-ku, Yokohama 223-8522, Japan
| | - Sho Tanosaki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
| | - Shogo Miyata
- Department of Mechanical Engineering, Faculty of Science and Technology, Keio University, 3-14-1 Kohoku-ku, Yokohama 223-8522, Japan.
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21
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MicroRNA profiling reveals important functions of miR-125b and let-7a during human retinal pigment epithelial cell differentiation. Exp Eye Res 2020; 190:107883. [DOI: 10.1016/j.exer.2019.107883] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 10/20/2019] [Accepted: 11/19/2019] [Indexed: 12/30/2022]
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22
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Karamali F, Esfahani MHN, Hajian M, Ejeian F, Satarian L, Baharvand H. Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells. J Cell Physiol 2019; 234:4256-4266. [PMID: 30191983 DOI: 10.1002/jcp.27194] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2018] [Accepted: 07/17/2018] [Indexed: 01/09/2023]
Abstract
Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.
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Affiliation(s)
- Fereshteh Karamali
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Mohammad-Hossein Nasr Esfahani
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
| | - Mehdi Hajian
- Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
| | - Fatemeh Ejeian
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
| | - Leila Satarian
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
- 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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23
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Vahdat S, Pahlavan S, Aghdami N, Bakhshandeh B, Baharvand H. Establishment of A Protocol for In Vitro Culture of Cardiogenic Mesodermal Cells Derived from Human Embryonic Stem Cells. CELL JOURNAL 2019; 20:496-504. [PMID: 30123995 PMCID: PMC6099148 DOI: 10.22074/cellj.2019.5661] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/09/2017] [Accepted: 02/19/2017] [Indexed: 11/11/2022]
Abstract
OBJECTIVE Cardiovascular progenitor cells (CPCs) are introduced as one of the promising cell sources for preclinical studies and regenerative medicine. One of the earliest type of CPCs is cardiogenic mesoderm cells (CMCs), which have the capability to generate all types of cardiac lineage derivatives. In order to benefit from CMCs, development of an efficient culture strategy is required. We aim to explore an optimized culture condition that uses human embryonic stem cell (hESC)-derived CMCs. MATERIALS AND METHODS In this experimental study, hESCs were expanded and induced toward cardiac lineage in a suspension culture. Mesoderm posterior 1-positive (MESP1+) CMCs were subjected to four different culture conditions: i. Suspension culture of CMC spheroids, ii. Adherent culture of CMC spheroids, iii. Adherent culture of single CMCs using gelatin, and iv. Adherent culture of single CMCs using Matrigel. RESULTS Although, we observed no substantial changes in the percentage of MESP1+ cells in different culture conditions, there were significantly higher viability and total cell numbers in CMCs cultured on Matrigel (condition iv) compared to the other groups. CMCs cultivated on Matrigel maintained their progenitor cell signature, which included the tendency for cardiogenic differentiation. CONCLUSION These results showed the efficacy of an adherent culture on Matrigel for hESC-derived CMCs, which would facilitate their use for future applications.
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Affiliation(s)
- Sadaf Vahdat
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
- 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
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Behnaz Bakhshandeh
- Department of Biotechnology, College of Science, University of Tehran, 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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24
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Simão D, Silva MM, Terrasso AP, Arez F, Sousa MFQ, Mehrjardi NZ, Šarić T, Gomes-Alves P, Raimundo N, Alves PM, Brito C. Recapitulation of Human Neural Microenvironment Signatures in iPSC-Derived NPC 3D Differentiation. Stem Cell Reports 2018; 11:552-564. [PMID: 30057262 PMCID: PMC6094163 DOI: 10.1016/j.stemcr.2018.06.020] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Revised: 06/25/2018] [Accepted: 06/27/2018] [Indexed: 02/05/2023] Open
Abstract
Brain microenvironment plays an important role in neurodevelopment and pathology, where the extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on heterologous matrices poorly resembling brain ECM. Here, we employed neurospheroids to address microenvironment remodeling during neural differentiation of human stem cells, without the confounding effects of exogenous matrices. Proteome and transcriptome dynamics revealed significant changes at cell membrane and ECM during 3D differentiation, diverging significantly from the 2D differentiation. Structural proteoglycans typical of brain ECM were enriched during 3D differentiation, in contrast to basement membrane constituents in 2D. Moreover, higher expression of synaptic and ion transport machinery was observed in 3D cultures, suggesting higher neuronal maturation in neurospheroids. This work demonstrates that 3D neural differentiation as neurospheroids promotes the expression of cellular and extracellular features found in neural tissue, highlighting its value to address molecular defects in cell-ECM interactions associated with neurological disorders.
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Affiliation(s)
- Daniel Simão
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Marta M Silva
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Ana P Terrasso
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Francisca Arez
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Marcos F Q Sousa
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Narges Z Mehrjardi
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Tomo Šarić
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Patrícia Gomes-Alves
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Nuno Raimundo
- Universitätsmedizin Göttingen, Institut für Zellbiochemie, Göttingen, Germany
| | - Paula M Alves
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Biológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
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25
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Shoji M, Minato H, Ogaki S, Seki M, Suzuki Y, Kume S, Kuzuhara T. Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells. PLoS One 2018; 13:e0201239. [PMID: 30048506 PMCID: PMC6062072 DOI: 10.1371/journal.pone.0201239] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Accepted: 07/11/2018] [Indexed: 12/19/2022] Open
Abstract
The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.
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Affiliation(s)
- Masaki Shoji
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
- * E-mail: (MS); (TK)
| | - Hiroki Minato
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
| | - Soichiro Ogaki
- School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan
| | - Masahide Seki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
| | - Yutaka Suzuki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
| | - Shoen Kume
- School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan
| | - Takashi Kuzuhara
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
- * E-mail: (MS); (TK)
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26
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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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27
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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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28
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Heidariyan Z, Ghanian MH, Ashjari M, Farzaneh Z, Najarasl M, Rezaei Larijani M, Piryaei A, Vosough M, Baharvand H. Efficient and cost-effective generation of hepatocyte-like cells through microparticle-mediated delivery of growth factors in a 3D culture of human pluripotent stem cells. Biomaterials 2018; 159:174-188. [DOI: 10.1016/j.biomaterials.2018.01.005] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2017] [Revised: 12/31/2017] [Accepted: 01/01/2018] [Indexed: 12/11/2022]
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29
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Farzaneh Z, Najarasl M, Abbasalizadeh S, Vosough M, Baharvand H. Developing a Cost-Effective and Scalable Production of Human Hepatic Competent Endoderm from Size-Controlled Pluripotent Stem Cell Aggregates. Stem Cells Dev 2018; 27:262-274. [PMID: 29298619 DOI: 10.1089/scd.2017.0074] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 μM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 μm; medium: 142 ± 32 μm; large: 214 ± 34 μm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 μM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.
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Affiliation(s)
- Zahra Farzaneh
- 1 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology , ACECR, Tehran, Iran
| | - Mostafa Najarasl
- 1 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology , ACECR, Tehran, Iran
| | - Saeed Abbasalizadeh
- 2 Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology , ACECR, Tehran, Iran
- 3 Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, Instituto Superior Técnico , Lisboa, Portugal
| | - Massoud Vosough
- 1 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology , ACECR, Tehran, Iran
| | - Hossein Baharvand
- 1 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology , ACECR, Tehran, Iran
- 4 Department of Developmental Biology, University of Science and Culture , Tehran, Iran
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30
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Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines. Eur J Cell Biol 2017; 96:347-355. [PMID: 28476334 DOI: 10.1016/j.ejcb.2017.03.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2016] [Revised: 02/25/2017] [Accepted: 03/28/2017] [Indexed: 02/02/2023] Open
Abstract
OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4, OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3, is located ∼9kb downstream of the E0 of OCT4C/C1. PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10-24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A, a slight upregulation of OCT4B1, and a dramatic upregulation of OCT4B. Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.
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31
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Scaling-Up Techniques for the Nanofabrication of Cell Culture Substrates via Two-Photon Polymerization for Industrial-Scale Expansion of Stem Cells. MATERIALS 2017; 10:ma10010066. [PMID: 28772424 PMCID: PMC5344595 DOI: 10.3390/ma10010066] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Revised: 01/05/2017] [Accepted: 01/10/2017] [Indexed: 01/28/2023]
Abstract
Stem-cell-based therapies require a high number (106–109) of cells, therefore in vitro expansion is needed because of the initially low amount of stem cells obtainable from human tissues. Standard protocols for stem cell expansion are currently based on chemically-defined culture media and animal-derived feeder-cell layers, which expose cells to additives and to xenogeneic compounds, resulting in potential issues when used in clinics. The two-photon laser polymerization technique enables three-dimensional micro-structures to be fabricated, which we named synthetic nichoids. Here we review our activity on the technological improvements in manufacturing biomimetic synthetic nichoids and, in particular on the optimization of the laser-material interaction to increase the patterned area and the percentage of cell culture surface covered by such synthetic nichoids, from a low initial value of 10% up to 88% with an optimized micromachining time. These results establish two-photon laser polymerization as a promising tool to fabricate substrates for stem cell expansion, without any chemical supplement and in feeder-free conditions for potential therapeutic uses.
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5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts. Sci Rep 2016; 6:37017. [PMID: 27841324 PMCID: PMC5107985 DOI: 10.1038/srep37017] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2016] [Accepted: 10/24/2016] [Indexed: 12/11/2022] Open
Abstract
Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier.
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AKT/GSK3β signaling pathway is critically involved in human pluripotent stem cell survival. Sci Rep 2016; 6:35660. [PMID: 27762303 PMCID: PMC5071844 DOI: 10.1038/srep35660] [Citation(s) in RCA: 59] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2015] [Accepted: 10/04/2016] [Indexed: 01/24/2023] Open
Abstract
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3β signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3β inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3β, and thus results relevant for PSC survival.
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Li B, Zhuang M, Wu C, Niu B, Zhang Z, Li X, Wei Z, Li G, Hua J. Bovine male germline stem-like cells cultured in serum- and feeder-free medium. Cytotechnology 2016; 68:2145-2157. [PMID: 26883918 PMCID: PMC5023554 DOI: 10.1007/s10616-015-9933-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2015] [Accepted: 11/14/2015] [Indexed: 12/19/2022] Open
Abstract
Male germline stem cells (mGSCs) presented in male testis are responsible for spermatogenesis during their whole life. However, little information can be found on the culture of bovine mGSCs, and the current culture system needs to be improved. In this study, we compared the effects of several commercial serum-free media and different extra-cellular matrix on the enrichment and cultivation of mGSCs. To find out the best culture condition, the biological characteristics of the cultured cells were evaluated by morphological observation, RT-PCR and immunofluorescent staining. According to the cells' condition in different experiment groups, we found out an efficient cultivation system for bovine mGSCs derived from neonate testis. In this serum- and feeder-free medium, the cultured cells maintained the typical morphology, and expressed specific surface markers of both pluripotent ES cells and mGSCs, including SSEA-1, CD49f, C-MYC, PLZF, GFRα1, LIN28, NANOG, Oct4 and SOX2 in commercial human ESCs medium PeproGrow-hESC + BIO (6-bromoindirubin-3'-oxime). Embryoid bodies, derived from the bovine mGSCs, and were formed by ganging drop culture. The retinoic acid induced bovine mGSCs were positive for Stra8, SCP3, DZAL, EMA1 and VASA, and resembled spermatid cells morphologically. Thus, we found an efficient bovine mGSCs-cultivation system, which is lack in serum and feeder.
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Affiliation(s)
- Bo Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Mengru Zhuang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Chongyang Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Bowen Niu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Zhou Zhang
- Department of Reproduction Centre, Shaanxi Provine Women and Children Hospital, Xi'an, 710000, Shaanxi Province, China
| | - Xin Li
- Department of Clinic Medicine, Bengbu Medicine University, Bengbu, Anhui, China
| | - Zhuying Wei
- Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China
| | - Guangpeng Li
- Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China.
| | - Jinlian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China.
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Zhang D, Mai Q, Li T, Huang J, Ding C, Jia M, Zhou C, Xu Y. Comparison of a xeno-free and serum-free culture system for human embryonic stem cells with conventional culture systems. Stem Cell Res Ther 2016; 7:101. [PMID: 27474011 PMCID: PMC4967296 DOI: 10.1186/s13287-016-0347-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2016] [Revised: 05/14/2016] [Accepted: 06/10/2016] [Indexed: 01/11/2023] Open
Abstract
BACKGROUND Elimination of all animal components during derivation and long-term culture of human embryonic stem cells (hESCs) is necessary for future applications of hESCs in clinical cell therapy. METHODS In this study, we established the culture system of xeno-free human foreskin fibroblast feeders (XF-HFF) in combination with chemically defined medium (CDM). XF-HFF/CDM was compared with several conventional culture systems. The hESCs cultured in different media were further characterized through karyotype analysis, pluripotency gene expression, and cell differentiation ability. RESULTS The hESCs in the XF-HFF/CDM maintained their characteristics including typical morphology and stable karyotype. In addition, hESCs were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. RT-PCR analysis shown that the stem cell markers OCT3/4, hTERT, SOX2, and Nanog were present in the cell line hESC-1 grown on XF-HFF/CDM. Furthermore, the results of cell growth and expression of bFGF, Oct-4, and hTERT indicated that XF-HFF/CDM had better performance than human serum-matrix/CDM and XF-HFF/human serum. CONCLUSION The comparison of different xeno-free culture conditions will facilitate clarifying the key features of self-renewal, pluripotency, and derivation and will shed light on clinic applications of hESCs.
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Affiliation(s)
- Dan Zhang
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Qingyun Mai
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Tao Li
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Jia Huang
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Chenhui Ding
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Mengxi Jia
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China
| | - Canquan Zhou
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China.
| | - Yanwen Xu
- Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, People's Republic of China.
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Adnan N, Mie M, Haque A, Hossain S, Mashimo Y, Akaike T, Kobatake E. Construction of a Defined Biomimetic Matrix for Long-Term Maintenance of Mouse Induced Pluripotent Stem Cells. Bioconjug Chem 2016; 27:1599-605. [PMID: 27269811 DOI: 10.1021/acs.bioconjchem.6b00141] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3β (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.
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Affiliation(s)
- Nihad Adnan
- Department of Environmental Chemistry and Engineering, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology , Yokohama, 226-8502, Japan
| | - Masayasu Mie
- Department of Environmental Chemistry and Engineering, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology , Yokohama, 226-8502, Japan
| | - Amranul Haque
- Department of Biomedical Engineering, University of California Davis , Davis, California 95616, United States
| | - Sharif Hossain
- Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of International Science , Tsukuba, 305-0821, Japan
| | - Yasumasa Mashimo
- Department of Environmental Chemistry and Engineering, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology , Yokohama, 226-8502, Japan
| | - Toshihiro Akaike
- Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of International Science , Tsukuba, 305-0821, Japan
| | - Eiry Kobatake
- Department of Environmental Chemistry and Engineering, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology , Yokohama, 226-8502, Japan
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Nakashima Y, Omasa T. What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium? Biores Open Access 2016; 5:84-93. [PMID: 27096107 PMCID: PMC4834485 DOI: 10.1089/biores.2016.0001] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.
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Affiliation(s)
- Yoshiki Nakashima
- Department of Material and Life Science, Graduate School of Engineering, Osaka University , Osaka, Japan
| | - Takeshi Omasa
- Department of Material and Life Science, Graduate School of Engineering, Osaka University , Osaka, Japan
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Rahimi Y, Mehdizadeh A, Nozad Charoudeh H, Nouri M, Valaei K, Fayezi S, Darabi M. Hepatocyte differentiation of human induced pluripotent stem cells is modulated by stearoyl-CoA desaturase 1 activity. Dev Growth Differ 2015; 57:667-74. [PMID: 26676854 DOI: 10.1111/dgd.12255] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Revised: 10/11/2015] [Accepted: 10/26/2015] [Indexed: 12/22/2022]
Abstract
Stearoyl-CoA desaturase 1 (SCD1) plays important roles in organ development, glucose tolerance, insulin sensitivity, and cancer. Here, we examined the role of SCD1 for the differentiation of human induced pluripotent stem (hiPS) cells to liver cells by using drug inhibition and biochemical experiments. hiPS cells cultured in a pro-hepatic medium were exposed to an SCD1 inhibitor at various stages throughout differentiation. Liver-specific markers, specifically α-fetoprotein, albumin and urea in conditioned medium, and hepatocyte nuclear factor 4α (HNF4α) and cytochrome P450 7A1 (CYP7A1) gene expressions and triglyceride in cellular extracts were analyzed at various development stages. Measures of hepatocyte-specific function and triglyceride accumulation in later stages were strongly inhibited a minimum of -29% (P < 0.05) by SCD1 inhibitor in the early stage of hepatic differentiation and effectively reversed (>30%, P < 0.01) by the addition of oleate. The results were also reproducible with human primary mononuclear cells (hPMN). SCD1 inhibitor had no significant effect on liver-specific markers when it was added in the hepatic maturation stage. However, it strikingly led to higher albumin (1.6-fold, P = 0.03) and urea (1.9-fold, P = 0.02) production, and HNF4α (1.9-fold, P = 0.02) and CYP7A1 (1.3-fold, P = 0.03) expression upon incubation during the lineage-commitment stage. Hepatic differentiation from cultured hiPS cells is sensitive to SCD1 inhibition and this sensitivity is affected by the stage of cellular differentiation. Notably, findings also indicate that this notion can be extended to hPMN. The requirement for SCD1 activity in functional differentiation of hepatocytes may have relevance for human liver disease and metabolic dysregulation.
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Affiliation(s)
- Yaghoub Rahimi
- Stem Cell Research Center, School of Medicine, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran.,Department of Biochemistry and Clinical Laboratories, School of Medicine, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
| | - Amir Mehdizadeh
- Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
| | - Hojjatollah Nozad Charoudeh
- Stem Cell Research Center, School of Medicine, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
| | - Mohammad Nouri
- Department of Biochemistry and Clinical Laboratories, School of Medicine, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
| | - Kobra Valaei
- Stem Cell Research Center, School of Medicine, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
| | - Shabnam Fayezi
- Students Research Committee, Department of Anatomy and Cell Biology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, 19989-99513, Iran
| | - Masoud Darabi
- Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences, Tabriz, 51666-15556, Iran
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Kajabadi NS, Ghoochani A, Peymani M, Ghaedi K, Kiani-Esfahani A, Hashemi MS, Nasr-Esfahani MH, Baharvand H. The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome Proliferative-activated Receptor-γ (PPARγ) Activation and Rho-associated Kinase (ROCK) Inhibition. J Biol Chem 2015; 290:26303-26313. [PMID: 26336103 PMCID: PMC4646278 DOI: 10.1074/jbc.m114.624841] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2014] [Revised: 08/25/2015] [Indexed: 01/26/2023] Open
Abstract
Although human pluripotent stem cells (hPSCs) provide valuable sources for regenerative medicine, their applicability is dependent on obtaining both suitable up-scaled and cost effective cultures. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSC survival upon dissociation; however, cloning efficiency is often still low. Here we have shown that pioglitazone, a selective peroxisome proliferative-activated receptor-γ agonist, along with Y-27632 synergistically diminished dissociation-induced apoptosis and increased cloning efficiency (2-3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous β-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptor-γ antagonist. This novel setting provided a step toward hPSC manipulation and its biomedical applications.
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Affiliation(s)
- Nasim-Sadat Kajabadi
- From the Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan 8174673441, Iran
| | - Ali Ghoochani
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran
| | - Maryam Peymani
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran
| | - Kamran Ghaedi
- From the Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan 8174673441, Iran, Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran,
| | - Abbas Kiani-Esfahani
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran
| | - Motahareh-Sadat Hashemi
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran
| | - Mohammad Hossein Nasr-Esfahani
- Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan 8165131378, Iran,
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran, and
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Du SH, Tay JCK, Chen C, Tay FC, Tan WK, Li ZD, Wang S. Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion. J Biosci Bioeng 2015; 120:210-7. [DOI: 10.1016/j.jbiosc.2014.12.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2014] [Revised: 11/19/2014] [Accepted: 12/08/2014] [Indexed: 12/15/2022]
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Assou S, Pourret E, Péquignot M, Rigau V, Kalatzis V, Aït-Ahmed O, Hamamah S. Cultured Cells from the Human Oocyte Cumulus Niche Are Efficient Feeders to Propagate Pluripotent Stem Cells. Stem Cells Dev 2015; 24:2317-27. [PMID: 26153797 DOI: 10.1089/scd.2015.0043] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Pluripotency is at the crossroads of stem cell research and biology of reproduction. The mature metaphase II oocyte contains the key factors for pluripotency induction and maintenance as assessed by its capacity to reprogram somatic nuclei. The cumulus cells (CCs) niche that surrounds the oocyte is crucial for its maturation and presumably for the oocyte to acquire its competence to confer pluripotency. In this study, we examined whether cells cultured from the human mature metaphase II oocyte CC niche (hCC) could be used as feeders for the propagation of human induced pluripotent stem cells. The induced pluripotent (iPS) cells cultured on hCC (hCC-iPS) were assessed for their pluripotency potential by their expression of pluripotency-associated genes such as Oct4, Nanog, and TRA1-60 and their competence to differentiate into the three germ layers in vitro (embryoid bodies) as well as in vivo (teratoma formation). We show that not only the hCC-iPS cells maintained their pluripotency potential, but they also exhibited much better self-renewal performance in terms of proliferation rate compared to the same cells cultured on human foreskin fibroblast (hFF) feeders (hFF-iPS). A comparative gene expression profile study of hCC and hFF revealed significant differences (P < 0.05) in expression of cellular matrix components and an upregulation in hCC of genes known to be important players in cell proliferation such as interleukin 6 gene (IL6).
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Affiliation(s)
- Said Assou
- 1 Université Montpellier , UFR de Médecine, Montpellier, France .,2 Institute for Regenerative Medicine and Biotherapy, INSERM U1203, CHRU Montpellier, Hôpital Saint-Eloi , Montpellier, France
| | - Emilie Pourret
- 2 Institute for Regenerative Medicine and Biotherapy, INSERM U1203, CHRU Montpellier, Hôpital Saint-Eloi , Montpellier, France
| | - Marie Péquignot
- 3 Institut des Neurosciences de Montpellier, INSERM U1051, Hôpital Saint-Eloi , Montpellier, France
| | - Valérie Rigau
- 4 Pathology Department, University Hospital Gui de Chauliac , Montpellier, France
| | - Vasiliki Kalatzis
- 3 Institut des Neurosciences de Montpellier, INSERM U1051, Hôpital Saint-Eloi , Montpellier, France
| | - Ounissa Aït-Ahmed
- 2 Institute for Regenerative Medicine and Biotherapy, INSERM U1203, CHRU Montpellier, Hôpital Saint-Eloi , Montpellier, France
| | - Samir Hamamah
- 1 Université Montpellier , UFR de Médecine, Montpellier, France .,2 Institute for Regenerative Medicine and Biotherapy, INSERM U1203, CHRU Montpellier, Hôpital Saint-Eloi , Montpellier, France .,5 ART-PGD Department, CHU Montpellier, Hôpital Arnaud de Villeneuve , Montpellier, France
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Seki T, Fukuda K. Methods of induced pluripotent stem cells for clinical application. World J Stem Cells 2015; 7:116-125. [PMID: 25621111 PMCID: PMC4300922 DOI: 10.4252/wjsc.v7.i1.116] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Revised: 09/18/2014] [Accepted: 10/27/2014] [Indexed: 02/06/2023] Open
Abstract
Reprograming somatic cells using exogenetic gene expression represents a groundbreaking step in regenerative medicine. Induced pluripotent stem cells (iPSCs) are expected to yield novel therapies with the potential to solve many issues involving incurable diseases. In particular, applying iPSCs clinically holds the promise of addressing the problems of immune rejection and ethics that have hampered the clinical applications of embryonic stem cells. However, as iPSC research has progressed, new problems have emerged that need to be solved before the routine clinical application of iPSCs can become established. In this review, we discuss the current technologies and future problems of human iPSC generation methods for clinical use.
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Courtot AM, Magniez A, Oudrhiri N, Féraud O, Bacci J, Gobbo E, Proust S, Turhan AG, Bennaceur-Griscelli A. Morphological analysis of human induced pluripotent stem cells during induced differentiation and reverse programming. Biores Open Access 2014; 3:206-16. [PMID: 25371857 PMCID: PMC4215385 DOI: 10.1089/biores.2014.0028] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming.
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Affiliation(s)
- Anne-Marie Courtot
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France . ; Université Paris Sud , Faculté de Médecine, Le Kremlin Bicêtre, France
| | - Aurélie Magniez
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France
| | - Noufissa Oudrhiri
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France . ; Service d'Hématologie Biologique APHP, Hôpital Paul Brousse , GHU Paris Sud Villejuif, France
| | - Olivier Féraud
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France
| | - Josette Bacci
- Service de Neurologie, Hôpital Bicêtre APHP , Le Kremlin Bicêtre, France
| | - Emilie Gobbo
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France
| | - Stéphanie Proust
- Service de Virologie, Hôpital Bicêtre APHP , Le Kremlin Bicêtre, France
| | - Ali G Turhan
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France . ; Université Paris Sud , Faculté de Médecine, Le Kremlin Bicêtre, France . ; Service d'Hématologie Biologique APHP, Hôpital Bicêtre , GHU Paris Sud, Le Kremlin Bicêtre, France
| | - Annelise Bennaceur-Griscelli
- Inserm U935, ES-TEAM Paris Sud, Ingestem , Villejuif, France . ; Université Paris Sud , Faculté de Médecine, Le Kremlin Bicêtre, France . ; Service d'Hématologie Biologique APHP, Hôpital Paul Brousse , GHU Paris Sud Villejuif, France
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44
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Chew LJ, DeBoy CA, Senatorov VV. Finding degrees of separation: experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting. J Neurosci Methods 2014; 236:125-47. [PMID: 25169049 PMCID: PMC4171043 DOI: 10.1016/j.jneumeth.2014.08.017] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2014] [Revised: 08/15/2014] [Accepted: 08/18/2014] [Indexed: 12/20/2022]
Abstract
The study of CNS glial cell function requires experimental methods to detect, purify, and manipulate each cell population with fidelity and specificity. With the identification and cloning of cell- and stage-specific markers, glial cell analysis techniques have grown beyond physical methods of tissue dissociation and cell culture, and become highly specific with immunoselection of cell cultures in vitro and genetic targeting in vivo. The unique plasticity of glial cells offers the potential for cell replacement therapies in neurological disease that utilize neural cells derived from transplanted neural stem and progenitor cells. In this mini-review, we outline general physical and genetic approaches for macroglial cell generation. We summarize cell culture methods to obtain astrocytes and oligodendrocytes and their precursors, from developing and adult tissue, as well as approaches to obtain human neural progenitor cells through the establishment of stem cells. We discuss popular targeting rodent strains designed for cell-specific detection, selection and manipulation of neuroglial cell progenitors and their committed progeny. Based on shared markers between astrocytes and stem cells, we discuss genetically modified mouse strains with overlapping expression, and highlight SOX-expressing strains available for targeting of stem and progenitor cell populations. We also include recently established mouse strains for detection, and tag-assisted RNA and miRNA analysis. This discussion aims to provide a brief overview of the rapidly expanding collection of experimental approaches and genetic resources for the isolation and targeting of macroglial cells, their sources, progeny and gene products to facilitate our understanding of their properties and potential application in pathology.
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Affiliation(s)
- Li-Jin Chew
- Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC, United States.
| | - Cynthia A DeBoy
- Biology Department, Trinity Washington University, Washington, DC, United States
| | - Vladimir V Senatorov
- Helen Wills Neuroscience Institute, University of California, Berkeley, CA, United States
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Oberwallner B, Brodarac A, Anić P, Šarić T, Wassilew K, Neef K, Choi YH, Stamm C. Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells. Eur J Cardiothorac Surg 2014; 47:416-25; discussion 425. [PMID: 24778452 DOI: 10.1093/ejcts/ezu163] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
OBJECTIVES Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology. RESULTS cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation. CONCLUSIONS Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.
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Affiliation(s)
- Barbara Oberwallner
- Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany
| | - Andreja Brodarac
- Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany
| | - Petra Anić
- Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany
| | - Tomo Šarić
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Center, University of Cologne, Cologne, Germany
| | | | - Klaus Neef
- Department of Cardiac and Thoracic Surgery, Heart Center, University of Cologne, Cologne, Germany
| | - Yeong-Hoon Choi
- Department of Cardiac and Thoracic Surgery, Heart Center, University of Cologne, Cologne, Germany
| | - Christof Stamm
- Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Campus Virchow-Klinikum, Berlin, Germany Deutsches Herzzentrum Berlin (DHZB), Berlin, Germany
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46
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Kalaskar VK, Lauderdale JD. Mouse embryonic development in a serum-free whole embryo culture system. J Vis Exp 2014. [PMID: 24637443 DOI: 10.3791/50803] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 °C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.
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Affiliation(s)
- Vijay K Kalaskar
- Neuroscience Division of the Biomedical & Health Sciences Institute, University of Georgia
| | - James D Lauderdale
- Neuroscience Division of the Biomedical & Health Sciences Institute, University of Georgia; Department of Cellular Biology, University of Georgia;
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47
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Ono T, Suzuki Y, Kato Y, Fujita R, Araki T, Yamashita T, Kato H, Torii R, Sato N. A single-cell and feeder-free culture system for monkey embryonic stem cells. PLoS One 2014; 9:e88346. [PMID: 24505480 PMCID: PMC3915054 DOI: 10.1371/journal.pone.0088346] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2013] [Accepted: 01/11/2014] [Indexed: 12/20/2022] Open
Abstract
Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
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Affiliation(s)
- Takashi Ono
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
- * E-mail:
| | - Yutaka Suzuki
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Yosuke Kato
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Risako Fujita
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Toshihiro Araki
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Tomoko Yamashita
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Hidemasa Kato
- Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan
| | - Ryuzo Torii
- Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Naoya Sato
- Discovery Molecular Pharmacology Department, Discovery Screening Center, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
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48
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Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, Toyoda T, Osafune K, Sekiguchi K, Yamanaka S. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 2014; 4:3594. [PMID: 24399248 PMCID: PMC3884228 DOI: 10.1038/srep03594] [Citation(s) in RCA: 454] [Impact Index Per Article: 41.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2013] [Accepted: 12/06/2013] [Indexed: 12/12/2022] Open
Abstract
In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.
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Affiliation(s)
- Masato Nakagawa
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | | | - Sho Senda
- Institute for Innovation, Ajinomoto CO., Inc., Kawasaki, 210-8681 Japan
| | - Nanako Takizawa
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Tomoko Ichisaka
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Kanako Asano
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Asuka Morizane
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Daisuke Doi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Jun Takahashi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Masatoshi Nishizawa
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Yoshinori Yoshida
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Taro Toyoda
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Kenji Osafune
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | | | - Shinya Yamanaka
- 1] Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan [2] Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158 USA
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Vosough M, Omidinia E, Kadivar M, Shokrgozar MA, Pournasr B, Aghdami N, Baharvand H. Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture. Stem Cells Dev 2013; 22:2693-2705. [PMID: 23731381 DOI: 10.1089/scd.2013.0088] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Recent advances in human embryonic and induced pluripotent stem cell-based therapies in animal models of hepatic failure have led to an increased appreciation of the need to translate the proof-of-principle concepts into more practical and feasible protocols for scale up and manufacturing of functional hepatocytes. In this study, we describe a scalable stirred-suspension bioreactor culture of functional hepatocyte-like cells (HLCs) from the human pluripotent stem cells (hPSCs). To promote the initial differentiation of hPSCs in a carrier-free suspension stirred bioreactor into definitive endoderm, we used rapamycin for "priming" phase and activin A for induction. The cells were further differentiated into HLCs in the same system. HLCs were characterized and then purified based on their physiological function, the uptake of DiI-acetylated low-density lipoprotein (LDL) by flow cytometry without genetic manipulation or antibody labeling. The sorted cells were transplanted into the spleens of mice with acute liver injury from carbon tetrachloride. The differentiated HLCs had multiple features of primary hepatocytes, for example, the expression patterns of liver-specific marker genes, albumin secretion, urea production, collagen synthesis, indocyanin green and LDL uptake, glycogen storage, and inducible cytochrome P450 activity. They increased the survival rate, engrafted successfully into the liver, and continued to present hepatic function (i.e., albumin secretion after implantation). This amenable scaling up and outlined enrichment strategy provides a new platform for generating functional HLCs. This integrated approach may facilitate biomedical applications of the hPSC-derived hepatocytes.
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Affiliation(s)
- Massoud Vosough
- 1 Department of Biochemistry, Pasteur Institute of Iran , Tehran, Iran
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50
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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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