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Xiong H, Lin B, Liu J, Lu R, Lin Z, Hang C, Liu W, Zhang L, Ding J, Guo H, Zhang M, Wang S, Gong Z, Xie D, Liu Y, Shi D, Liang D, Liu Z, Chen YH, Yang J. SALL2 regulates neural differentiation of mouse embryonic stem cells through Tuba1a. Cell Death Dis 2024; 15:710. [PMID: 39349437 PMCID: PMC11442768 DOI: 10.1038/s41419-024-07088-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 09/11/2024] [Accepted: 09/16/2024] [Indexed: 10/02/2024]
Abstract
The spalt (Sal) gene family has four members (Sall1-4) in vertebrates, all of which play pivotal roles in various biological processes and diseases. However, the expression and function of SALL2 in development are still less clear. Here, we first charted SALL2 protein expression pattern during mouse embryo development by immunofluorescence, which revealed its dominant expression in the developing nervous system. With the establishment of Sall2 deficient mouse embryonic stem cells (ESCs), the in vitro neural differentiation system was leveraged to interrogate the function of SALL2, which showed impaired neural differentiation of Sall2 knockout (KO) ESCs. Furthermore, neural stem cells (NSCs) could not be derived from Sall2 KO ESCs and the generation of neural tube organoids (NTOs) was greatly inhibited in the absence of SALL2. Meanwhile, transgenic expression of E1 isoform of SALL2 restored the defects of neural differentiation in Sall2 KO ESCs. By chromatin immunoprecipitation sequencing (ChIP-seq), Tuba1a was identified as downstream target of SALL2, whose function in neural differentiation was confirmed by rescuing neural phenotypes of Sall2 KO ESCs when overexpressed. In sum, by elucidating SALL2 expression dynamics during early mouse development and mechanistically characterizing its indispensable role in neural differentiation, this study offers insights into SALL2's function in human nervous system development, associated pathologies stemming from its mutations and relevant therapeutic strategy.
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Affiliation(s)
- Hui Xiong
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Department of Cell Biology, School of Medicine, Tongji University, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
| | - Bowen Lin
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Junyang Liu
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Department of Cell Biology, School of Medicine, Tongji University, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
| | - Renhong Lu
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Zheyi Lin
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Chengwen Hang
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Wenjun Liu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Lei Zhang
- Department of Anatomy, Histology and Embryology, School of Medicine, Tongji University, Shanghai, 200092, China
- Clinical Center for Brain and Spinal Cord Research, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Jie Ding
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Huixin Guo
- Department of Cardiology, the Second Hospital of Shanxi Medical University, Taiyuan, 030001, China
| | - Mingshuai Zhang
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Department of Cell Biology, School of Medicine, Tongji University, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
| | - Siyu Wang
- Jinzhou Medical University, Jinzhou, Liaoning, 121000, China
| | - Zheng Gong
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Jinzhou Medical University, Jinzhou, Liaoning, 121000, China
| | - Duanyang Xie
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Yi Liu
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
| | - Dan Shi
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
| | - Dandan Liang
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Shanghai, 200092, China
| | - Zhen Liu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yi-Han Chen
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China.
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China.
- Department of Pathology and Pathophysiology, School of Medicine, Tongji University, Shanghai, 200092, China.
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Shanghai, 200092, China.
| | - Jian Yang
- State Key Laboratory of Cardiovascular Diseases, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Shanghai Arrhythmia Research Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.
- Shanghai Frontiers Center of Nanocatalytic Medicine, Shanghai, 200092, China.
- Department of Cell Biology, School of Medicine, Tongji University, Shanghai, 200092, China.
- Clinical Center for Heart Research, Tongji University, Shanghai, 200092, China.
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Shanghai, 200092, China.
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Mensah IK, Gowher H. Signaling Pathways Governing Cardiomyocyte Differentiation. Genes (Basel) 2024; 15:798. [PMID: 38927734 PMCID: PMC11202427 DOI: 10.3390/genes15060798] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2024] [Revised: 06/13/2024] [Accepted: 06/13/2024] [Indexed: 06/28/2024] Open
Abstract
Cardiomyocytes are the largest cell type that make up the heart and confer beating activity to the heart. The proper differentiation of cardiomyocytes relies on the efficient transmission and perception of differentiation cues from several signaling pathways that influence cardiomyocyte-specific gene expression programs. Signaling pathways also mediate intercellular communications to promote proper cardiomyocyte differentiation. We have reviewed the major signaling pathways involved in cardiomyocyte differentiation, including the BMP, Notch, sonic hedgehog, Hippo, and Wnt signaling pathways. Additionally, we highlight the differences between different cardiomyocyte cell lines and the use of these signaling pathways in the differentiation of cardiomyocytes from stem cells. Finally, we conclude by discussing open questions and current gaps in knowledge about the in vitro differentiation of cardiomyocytes and propose new avenues of research to fill those gaps.
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Affiliation(s)
| | - Humaira Gowher
- Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
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3
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Fang J, Yang Q, Maas RGC, Buono M, Meijlink B, Lotgerink Bruinenberg D, Benavente ED, Mokry M, van Mil A, Qian L, Goumans MJ, Schiffelers R, Lei Z, Sluijter JPG. Vitamin C facilitates direct cardiac reprogramming by inhibiting reactive oxygen species. Stem Cell Res Ther 2024; 15:19. [PMID: 38229180 PMCID: PMC10792814 DOI: 10.1186/s13287-023-03615-x] [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: 06/08/2023] [Accepted: 12/14/2023] [Indexed: 01/18/2024] Open
Abstract
BACKGROUND After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.
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Affiliation(s)
- Juntao Fang
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
- Circulatory Health Laboratory, UMC Utrecht, Regenerative Medicine Center Utrecht, University Utrecht, 3508 GA, Utrecht, The Netherlands
| | - Qiangbing Yang
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
- CDL Research, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Renée G C Maas
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
- Circulatory Health Laboratory, UMC Utrecht, Regenerative Medicine Center Utrecht, University Utrecht, 3508 GA, Utrecht, The Netherlands
| | - Michele Buono
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Bram Meijlink
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Dyonne Lotgerink Bruinenberg
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Ernest Diez Benavente
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Michal Mokry
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
- CDL Research, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Alain van Mil
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands
- Circulatory Health Laboratory, UMC Utrecht, Regenerative Medicine Center Utrecht, University Utrecht, 3508 GA, Utrecht, The Netherlands
| | - Li Qian
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, USA
| | - Marie-José Goumans
- Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands
| | | | - Zhiyong Lei
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
- CDL Research, University Medical Center Utrecht, Utrecht, The Netherlands.
- Circulatory Health Laboratory, UMC Utrecht, Regenerative Medicine Center Utrecht, University Utrecht, 3508 GA, Utrecht, The Netherlands.
| | - Joost P G Sluijter
- Experimental Cardiology laboratory, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
- Circulatory Health Laboratory, UMC Utrecht, Regenerative Medicine Center Utrecht, University Utrecht, 3508 GA, Utrecht, The Netherlands.
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Martins GLS, Nonaka CKV, Rossi EA, de Lima AVR, Adanho CSA, Oliveira MS, Yahouedehou SCMA, de Souza CLEM, Gonçalves MDS, Paredes BD, Souza BSDF. Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells. Cells 2023; 12:cells12081121. [PMID: 37190030 DOI: 10.3390/cells12081121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2022] [Revised: 03/13/2023] [Accepted: 03/30/2023] [Indexed: 05/17/2023] Open
Abstract
BACKGROUND Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. METHODS iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. RESULTS Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. CONCLUSION A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.
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Affiliation(s)
- Gabriele Louise Soares Martins
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
| | - Carolina Kymie Vasques Nonaka
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
- D'Or Institute for Research and Education (IDOR), Salvador 41253-190, Brazil
| | - Erik Aranha Rossi
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
| | - Adne Vitória Rocha de Lima
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
| | - Corynne Stephanie Ahouefa Adanho
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
| | - Moisés Santana Oliveira
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
| | | | | | | | - Bruno Diaz Paredes
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
- D'Or Institute for Research and Education (IDOR), Salvador 41253-190, Brazil
| | - Bruno Solano de Freitas Souza
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil
- Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil
- D'Or Institute for Research and Education (IDOR), Salvador 41253-190, Brazil
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Choi D, Gwon K, Hong HJ, Baskaran H, Calvo-Lozano O, Gonzalez-Suarez AM, Park K, de Hoyos-Vega JM, Lechuga LM, Hong J, Stybayeva G, Revzin A. Coating Bioactive Microcapsules with Tannic Acid Enhances the Phenotype of the Encapsulated Pluripotent Stem Cells. ACS APPLIED MATERIALS & INTERFACES 2022; 14:10.1021/acsami.2c06783. [PMID: 35658394 PMCID: PMC10314364 DOI: 10.1021/acsami.2c06783] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Human pluripotent stem cells (hPSCs) may be differentiated into any adult cell type and therefore hold incredible promise for cell therapeutics and disease modeling. There is increasing interest in three-dimensional (3D) hPSC culture because of improved differentiation outcomes and potential for scale up. Our team has recently described bioactive heparin (Hep)-containing core-shell microcapsules that promote rapid aggregation of stem cells into spheroids and may also be loaded with growth factors for the local and sustained delivery to the encapsulated cells. In this study, we explored the possibility of further modulating bioactivity of microcapsules through the use of an ultrathin coating composed of tannic acid (TA). Deposition of the TA film onto model substrates functionalized with Hep and poly(ethylene glycol) was characterized by ellipsometry and atomic force microscopy. Furthermore, the presence of the TA coating was observed to increase the amount of basic fibroblast growth factor (bFGF) incorporation by up to twofold and to extend its release from 5 to 7 days. Most significantly, TA-microcapsules loaded with bFGF induced higher levels of pluripotency expression compared to uncoated microcapsules containing bFGF. Engineered microcapsules described here represent a new stem cell culture approach that enables 3D cultivation and relies on local delivery of inductive cues.
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Affiliation(s)
- Daheui Choi
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Kihak Gwon
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Hye Jin Hong
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Harihara Baskaran
- Department of Chemical and Biomolecular Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Olalla Calvo-Lozano
- Nanobiosensors and Bioanalytical Applications Group (NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, CIBERBBN and BIST, Barcelona 08193, Spain
| | - Alan M Gonzalez-Suarez
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Kyungtae Park
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 03722, Republic of Korea
| | - Jose M de Hoyos-Vega
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Laura M Lechuga
- Nanobiosensors and Bioanalytical Applications Group (NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, CIBERBBN and BIST, Barcelona 08193, Spain
| | - Jinkee Hong
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 03722, Republic of Korea
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, United States
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Weatherbee BAT, Cui T, Zernicka-Goetz M. Modeling human embryo development with embryonic and extra-embryonic stem cells. Dev Biol 2021; 474:91-99. [PMID: 33333069 PMCID: PMC8232073 DOI: 10.1016/j.ydbio.2020.12.010] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Revised: 12/09/2020] [Accepted: 12/10/2020] [Indexed: 12/18/2022]
Abstract
Early human post-implantation development involves extensive growth combined with a series of complex morphogenetic events. The lack of precise spatial and temporal control over these processes leads to pregnancy loss. Given the ethical and technical limitations in studying the natural human embryo, alternative approaches are needed to investigate mechanisms underlying this critical stage of human development. Here, we present an overview of the different stem cells and stem cell-derived models which serve as useful, albeit imperfect, tools in understanding human embryogenesis. Current models include stem cells that represent each of the three earliest lineages: human embryonic stem cells corresponding to the epiblast, hypoblast-like stem cells and trophoblast stem cells. We also review the use of human embryonic stem cells to model complex aspects of epiblast morphogenesis and differentiation. Additionally, we propose that the combination of both embryonic and extra-embryonic stem cells to form three-dimensional embryo models will provide valuable insights into cell-cell chemical and mechanical interactions that are essential for natural embryogenesis.
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Affiliation(s)
- Bailey A T Weatherbee
- Mouse and Human Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK
| | - Tongtong Cui
- Plasticity and Synthetic Embryology Group, California Institute of Technology, Division of Biology and Biological Engineering, 1200 E. California Boulevard, Pasadena, CA, 91125, USA
| | - Magdalena Zernicka-Goetz
- Mouse and Human Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK; Plasticity and Synthetic Embryology Group, California Institute of Technology, Division of Biology and Biological Engineering, 1200 E. California Boulevard, Pasadena, CA, 91125, USA.
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7
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Skowron MA, Watolla MM, Nettersheim D. Three-Dimensional Cultivation of Germ Cell Cancer Cell Lines as Hanging Drops. Methods Mol Biol 2021; 2195:77-83. [PMID: 32852758 DOI: 10.1007/978-1-0716-0860-9_6] [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] [Indexed: 02/18/2023]
Abstract
The hanging drop cell culture technique allows to study three-dimensional growth and differentiation of cell aggregates, that is, embryonic stem cells. Compared to standard two-dimensional monolayer cell cultivation, hanging drops allow for a better visualization and understanding of the developmental processes in vitro. Hanging drop cultivation can also be used to study biology of cancer cells three-dimensionally in vitro. This method can serve as an intermediate between the two-dimensional monolayer cell culture and in vivo models, which can be simply established in laboratories exhibiting minimum requirements of cell culture equipment. In this chapter, we describe the three-dimensional cultivation of germ cell cancer cell lines in hanging drops.
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Affiliation(s)
- Margaretha A Skowron
- Department of Urology, Urological Research Laboratory, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany
| | - Meike M Watolla
- Department of Urology, Urological Research Laboratory, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany
| | - Daniel Nettersheim
- Department of Urology, Urological Research Laboratory, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.
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8
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Park Y, Huh KM, Kang SW. Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research. Int J Mol Sci 2021; 22:2491. [PMID: 33801273 PMCID: PMC7958286 DOI: 10.3390/ijms22052491] [Citation(s) in RCA: 65] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2020] [Revised: 02/25/2021] [Accepted: 02/25/2021] [Indexed: 01/10/2023] Open
Abstract
The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.
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Affiliation(s)
- Yujin Park
- Department of Polymer Science and Engineering & Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, Korea;
- Predictive Model Research Center, Korea Institute of Toxicology, Daejeon 34114, Korea
| | - Kang Moo Huh
- Department of Polymer Science and Engineering & Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, Korea;
| | - Sun-Woong Kang
- Predictive Model Research Center, Korea Institute of Toxicology, Daejeon 34114, Korea
- Human and Environmental Toxicology Program, University of Science and Technology, Daejeon 34114, Korea
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9
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Markmee R, Aungsuchawan S, Tancharoen W, Narakornsak S, Pothacharoen P. Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine. Heliyon 2020; 6:e04844. [PMID: 32995593 PMCID: PMC7502343 DOI: 10.1016/j.heliyon.2020.e04844] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2020] [Revised: 05/01/2020] [Accepted: 09/01/2020] [Indexed: 12/18/2022] Open
Abstract
Human amniotic fluid mesenchymal stem cells (hAF-MSCs) have been shown to be effective in the treatment of many diseases. Platelet lysate (PL) contains multiple growth and differentiation factors; therefore, it can be used as a differentiation inducer. In this study, we attempted to evaluate the efficiency of human platelet lysate (hPL) on cell viability and the effects on cardiomyogenic differentiation of hAF-MSCs. When treating the cells with hPL, the result showed an increase in cell viability. Expressions of cardiomyogenic specific genes, including GATA4, cTnT, Cx43 and Nkx2.5, were higher in the combined treatment groups of 5-azacytidine (5-aza) and hPL than the expressions of cardiomyogenic specific genes in the control group and in the 5-aza treatment group. In terms of the results of immunofluorescence and immunoenzymatic staining, the highest expressions of cardiomyogenic specific proteins were revealed in combined treatment groups. It can be summarized that hPL may be an effective supporting cardiomyogenic supplementary factor for cardiomyogenic differentiation in hAF-MSCs.
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Affiliation(s)
- Runchana Markmee
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Sirinda Aungsuchawan
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Waleephan Tancharoen
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Suteera Narakornsak
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Peraphan Pothacharoen
- Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
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10
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Chen ACH, Lee KF, Yeung WSB, Lee YL. Human embryonic stem cells as an in vitro model for studying developmental origins of type 2 diabetes. World J Stem Cells 2020; 12:761-775. [PMID: 32952857 PMCID: PMC7477660 DOI: 10.4252/wjsc.v12.i8.761] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Revised: 04/28/2020] [Accepted: 06/14/2020] [Indexed: 02/06/2023] Open
Abstract
The developmental origins of health and diseases (DOHaD) is a concept stating that adverse intrauterine environments contribute to the health risks of offspring. Since the theory emerged more than 30 years ago, many epidemiological and animal studies have confirmed that in utero exposure to environmental insults, including hyperglycemia and chemicals, increased the risk of developing noncommunicable diseases (NCDs). These NCDs include metabolic syndrome, type 2 diabetes, and complications such as diabetic cardiomyopathy. Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development. Embryonic stem cells (ESCs) have also been utilized by researchers to study the DOHaD. ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage; therefore, they are excellent in vitro models for studying early developmental events. More importantly, human ESCs (hESCs) are the best alternative to human embryos for research because of ethical concerns. In this review, we will discuss different maternal conditions associated with DOHaD, focusing on the complications of maternal diabetes. Next, we will review the differentiation protocols developed to generate different cell lineages from hESCs. Additionally, we will review how hESCs are utilized as a model for research into the DOHaD. The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.
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Affiliation(s)
- Andy Chun-Hang Chen
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - Kai Fai Lee
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - William Shu Biu Yeung
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - Yin Lau Lee
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China.
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11
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Ali SR, Ahmad W, Naeem N, Salim A, Khan I. Small molecule 2'-deoxycytidine differentiates human umbilical cord-derived MSCs into cardiac progenitors in vitro and their in vivo xeno-transplantation improves cardiac function. Mol Cell Biochem 2020; 470:99-113. [PMID: 32415417 DOI: 10.1007/s11010-020-03750-6] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Accepted: 05/08/2020] [Indexed: 12/11/2022]
Abstract
Small molecules are widely used to induce stem cell differentiation. 2'-deoxycytidine (2-DC) belongs to the cytidine family. It stimulates the expression of cardiac-specific genes and proteins, and directs mesenchymal stem cells towards cardiomyogenic differentiation. We aim to investigate the role of 2-DC-treated human umbilical cord mesenchymal stem cells (UC-MSCs) into myogenic lineage and explore their application in regeneration of infarcted myocardium. UC-MSCs were treated with 5, 10, 20, and 40 µM 2-DC following optimization by cytotoxicity analysis. Rat model of myocardial infarction (MI) was induced by ligating left anterior descending coronary artery. Normal, and 2-DC treated UC-MSCs were transplanted in the left ventricular wall immediately after ligation. Echocardiographic measurements were performed to assess cardiac function. Tissue architecture of the myocardium was examined by histological analysis to determine fate of the transplanted cells. MSCs were successfully isolated from human umbilical cord tissue. 2-DC treatment did not produce any significant cytotoxic effect in UC-MSCs at all concentrations. qPCR analysis of treated UC-MSCs showed induction of myogenic differentiation, which is more pronounced at 20 μM concentration. Fluorescently labeled 2-DC-treated UC-MSCs showed significant (**P < 0.01) homing in the infarcted myocardium as compared to normal UC-MSCs. Hearts transplanted with 2-DC-treated UC-MSCs significantly (***P < 0.001) improved the cardiac systolic and diastolic functions and pumping ability as compared to normal UC-MSCs and MI groups. Fibrotic area and left ventricular wall thickness were significantly improved (***P < 0.001) in 2-DC-treated group as compared to normal UC-MSCs. Immunohistochemical staining showed co-localization of fluorescently labeled cells and patches of differentiated myocytes which were stained for cardiac proteins in the infarct zone implying that the treated UC-MSCs regenerated cardiomyocytes. We report for the first time that 2-DC induces cardiac differentiation in UC-MSCs. Transplanted cells differentiated into functional cardiomyocytes and significantly improved cardiac performance. These pre-differentiated cardiac progenitors showed better survival, homing, and distribution in the infarcted zone. 2-DC treated cells not only improved cardiac function, but also restored tissue homeostasis, suggesting a better therapeutic option for the regeneration of cardiac tissue in the clinical setup.
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Affiliation(s)
- Syeda Roohina Ali
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
| | - Waqas Ahmad
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
| | - Nadia Naeem
- Dow University of Health Sciences, Ojha Campus, Gulzar-e-Hijri, Suparco Road, KDA Scheme-33, Karachi, Pakistan
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.
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12
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Laco F, Lam ATL, Woo TL, Tong G, Ho V, Soong PL, Grishina E, Lin KH, Reuveny S, Oh SKW. Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor. Stem Cell Res Ther 2020; 11:118. [PMID: 32183888 PMCID: PMC7076930 DOI: 10.1186/s13287-020-01618-6] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 02/11/2020] [Accepted: 02/24/2020] [Indexed: 01/13/2023] Open
Abstract
Background The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Methods Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Results Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. Conclusion A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry. Supplementary information The online version of this article (10.1186/s13287-020-01618-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Filip Laco
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Alan Tin-Lun Lam
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
| | - Tsung-Liang Woo
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Gerine Tong
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Valerie Ho
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Poh-Loong Soong
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Elina Grishina
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Kun-Han Lin
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Shaul Reuveny
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Steve Kah-Weng Oh
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
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13
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Nakao S, Tsukamoto T, Ueyama T, Kawamura T. STAT3 for Cardiac Regenerative Medicine: Involvement in Stem Cell Biology, Pathophysiology, and Bioengineering. Int J Mol Sci 2020; 21:ijms21061937. [PMID: 32178385 PMCID: PMC7139789 DOI: 10.3390/ijms21061937] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Revised: 03/07/2020] [Accepted: 03/10/2020] [Indexed: 12/28/2022] Open
Abstract
Heart disease is the most common cause of death in developed countries, but the medical treatments for heart failure remain limited. In this context, the development of cardiac regeneration therapy for severe heart failure is important. Owing to their unique characteristics, including multiple differentiation and infinitive self-renewal, pluripotent stem cells can be considered as a novel source for regenerative medicine. Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling plays critical roles in the induction, maintenance, and differentiation of pluripotent stem cells. In the heart, JAK/STAT3 signaling has diverse cellular functions, including myocardial differentiation, cell cycle re-entry of matured myocyte after injury, and anti-apoptosis in pathological conditions. Therefore, regulating STAT3 activity has great potential as a strategy of cardiac regeneration therapy. In this review, we summarize the current understanding of STAT3, focusing on stem cell biology and pathophysiology, as they contribute to cardiac regeneration therapy. We also introduce a recently reported therapeutic strategy for myocardial regeneration that uses engineered artificial receptors that trigger endogenous STAT3 signal activation.
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Affiliation(s)
- Shu Nakao
- Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan; (S.N.); (T.T.); (T.U.)
- Ritsumeikan Global Innovation Research Institute, Ritsumeikan University, Kusatsu 525-8577, Japan
| | - Tasuku Tsukamoto
- Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan; (S.N.); (T.T.); (T.U.)
- Ritsumeikan Global Innovation Research Institute, Ritsumeikan University, Kusatsu 525-8577, Japan
| | - Tomoe Ueyama
- Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan; (S.N.); (T.T.); (T.U.)
- Ritsumeikan Global Innovation Research Institute, Ritsumeikan University, Kusatsu 525-8577, Japan
| | - Teruhisa Kawamura
- Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan; (S.N.); (T.T.); (T.U.)
- Ritsumeikan Global Innovation Research Institute, Ritsumeikan University, Kusatsu 525-8577, Japan
- Correspondence: ; Tel.: +81-75-599-4327
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14
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Bagheri-Hosseinabadi Z, Seyedi F, Mollaei HR, Moshrefi M, Seifalian A. Combination of 5-azaytidine and hanging drop culture convert fat cell into cardiac cell. Biotechnol Appl Biochem 2020; 68:92-101. [PMID: 32028539 DOI: 10.1002/bab.1897] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Accepted: 01/29/2020] [Indexed: 12/12/2022]
Abstract
One of the promising approaches for the treatment of cardiac disease is stem cell therapy. In this study, we compared the cardiomyogenic differentiation rate, from human adipose-derived stem cells (hADSCs) in a three-dimensional (3D) hanging drop (HD) spheroid culture system, versus a two-dimensional (2D) culture condition at different concentrations of 5-azacytidine (5-Aza). 5-Azaytidine (5-Aza) is a pyrimidine nucleoside analogue of cytidine that initiates cell differentiation programs through DNA demethylation. The hADSCs were isolated and cultured both in 2D and 3D HD conditions, with either 10 or 50 μM concentrations of 5-Aza. Then DNA content, gene expression, and protein content were analyzed. 3D HD culture resulted in a higher percentage of cells in G0/G1 and S phase in the cell division cycle, whereas 2D culture led to a greater percentage of cells in the G2/M phase. A significantly higher gene expression rate of HAND1, HAND2, cTnI, Cx43, βMHC, GATA4, NKX2.5, and MLC2V was observed in HD treated with 50 μM 5-Aza. This was confirmed by immunocytochemistry. These findings suggest that 50 μM concentration of 5-Aza can induce hADSCs to differentiate into cardiomyocytes. The differentiation rate was significantly higher when accompanied by the 3D HD culture system. This work provides a new culture system for cell differentiation for cardiovascular tissue engineering.
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Affiliation(s)
- Zahra Bagheri-Hosseinabadi
- Department of Clinical Biochemistry, Faculty of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.,Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
| | - Fatemeh Seyedi
- Department of Anatomy, School of Medicine, Jiroft University of Medical Sciences, Jiroft, Iran
| | - Hamid Reza Mollaei
- Department of Medical Microbiology, Afzalipour Medical Faculty, Kerman University of Medical Science, Kerman, Iran
| | - Mojgan Moshrefi
- Medical Nanotechnology & Tissue Engineering Research Centre, Yazd Reproductive Science Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
| | - Alexander Seifalian
- Nanotechnology and Regenerative Medicine Commercialisation Centre (NanoRegMed Ltd.), London BioScience Innovation Centre, London, United Kingdom
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15
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Rachel K, Pathak S, Moorthi A, Narasimhan S, Murugesan R, Narayan S. 5-Azacytidine incorporated polycaprolactone-gelatin nanoscaffold as a potential material for cardiomyocyte differentiation. JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 2019; 31:123-140. [DOI: 10.1080/09205063.2019.1678796] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Affiliation(s)
- Kerena Rachel
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
| | - Surajit Pathak
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
| | - A. Moorthi
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
| | - Srinivasan Narasimhan
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
| | - Ramachandran Murugesan
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
| | - Shoba Narayan
- Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamilnadu, India
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16
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Joshi J, Brennan D, Beachley V, Kothapalli CR. Cardiomyogenic differentiation of human bone marrow-derived mesenchymal stem cell spheroids within electrospun collagen nanofiber mats. J Biomed Mater Res A 2018; 106:3303-3312. [PMID: 30242963 DOI: 10.1002/jbm.a.36530] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Revised: 07/26/2018] [Accepted: 08/16/2018] [Indexed: 12/18/2022]
Abstract
Collagen is the major structural protein in myocardium and contributes to tissue strength and integrity, cellular orientation, and cell-cell and cell-matrix interactions. Significant post-myocardial infarction related loss of cardiomyocytes and cardiac tissue, and their subsequent replacement with fibrous scar tissue, negatively impacts endogenous tissue repair and regeneration capabilities. To overcome such limitations, tissue engineers are working toward developing a 3D cardiac patch which not only mimics the structural, functional, and biological hierarchy of the native cardiac tissue, but also could deliver autologous stem cells and encourage their homing and differentiation. In this study, we examined the utility of electrospun, randomly-oriented, type-I collagen nanofiber (dia = 789 ± 162 nm) mats on the cardiomyogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSC) spheroids, in the presence or absence of 10 μM 5-azacytidine (aza). Results showed that these scaffolds are biocompatible and enable time-dependent evolution of early (GATA binding protein 4: GATA4), late (cardiac troponin I: cTnI), and mature (myosin heavy chain: MHC) cardiomyogenic markers, with a simultaneous reduction in CD90 (stemness) expression, independent of aza-treatment. Aza-exposure improved connexin-4 expression and sustained sarcomeric α-actin expression, but provided only transient improvement in cardiac troponin T (cTnT) expression. Cell orientation and alignment significantly improved in these nanofiber scaffolds over time and with aza-exposure. Although further quantitative in vitro and in vivo studies are needed to establish the clinical applicability of such stem-cell laden collagen nanofiber mats as cardiac patches for cardiac tissue regeneration, our results underscore the benefits of 3D milieu provided by electrospun collagen nanofiber mats, aza, and spheroids on the survival, cardiac differentiation and maturation of human BM-MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3303-3312, 2018.
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Affiliation(s)
- Jyotsna Joshi
- Department of Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio, 44115
| | - David Brennan
- Department of Biomedical Engineering, Rowan University, Glassboro, New Jersey, 08028
| | - Vince Beachley
- Department of Biomedical Engineering, Rowan University, Glassboro, New Jersey, 08028
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17
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Khaksar M, Sayyari M, Rezaie J, Pouyafar A, Montazersaheb S, Rahbarghazi R. High glucose condition limited the angiogenic/cardiogenic capacity of murine cardiac progenitor cells in in vitro and in vivo milieu. Cell Biochem Funct 2018; 36:346-356. [PMID: 30051492 DOI: 10.1002/cbf.3354] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2018] [Revised: 06/19/2018] [Accepted: 07/02/2018] [Indexed: 12/21/2022]
Abstract
Murine c-kit+ cardiac cells were isolated and enriched by magnetic activated cell sorting technique. c-kit+ cells viability and colony-forming activity were evaluated by MTT and clonogenic assay. c-kit+ cells were exposed to endothelial, pericyte, and cardiomyocyte induction media containing 30mM glucose for 7 days. We monitored the level of endothelial (VE-cadherin, CD31, and vWF), pericyte (NG2 , α-SMA, and PDGFR-β), and cardiomyocyte markers (cTnT) using flow cytometry, real-time Polymerase Chain Reaction (PCR), and Enzyme-Linked Immunosorbent Assay (ELISA) analyses. Ultrastructural changes were studied by transmission electron microscopy (TEM) in cells treated with 5-Azacytidine and 30mM glucose. Matrigel plug assay was performed to determine the angio/cardiogenic property of c-kit+ cells in a diabetic mouse model. Glucose of 30mM decreased c-kit+ cells viability and clonogenicity (P < 0.05). The transdifferentiation capacity of c-kit+ cells into the endothelial lineage, pericytes, and cardiomyocytes were reduced through the inhibition of related genes (P < 0.05). TEM analysis revealed cardiomyocyte differentiation rate in c-kit+ cells coincided with an increased intracellular lipid accumulation and reduced number of mitochondria. Similar to in vitro condition, the angiogenic capacity of c-kit+ cells was aborted in vivo indicated by reduced NG2 , α-SMA, CD31, and vWF levels. High glucose condition reduces the angio/cardiogenic capacity of cardiac c-kit+ cells in vitro and in vivo. SIGNIFICANCE OF THE STUDY: High glucose condition seen in diabetes mellitus could affect the regenerative potential of cardiac tissue. The current experiment showed that the exposure of murine cardiac progenitor cells (CD117+ cells) to condition containing 30mM glucose could decrease the differentiation properties into endothelial cells, pericytes, and mature cardiomyocytes in vitro and in vivo. Our finding confirmed that the angiogenic/cardiogenic potential cardiac progenitor cells decrease under treatment with high glucose content as seen in the diabetic condition.
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Affiliation(s)
- Majid Khaksar
- Department of Pathology, Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran.,Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mansour Sayyari
- Department of Pathology, Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran
| | - Jafar Rezaie
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ayda Pouyafar
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Reza Rahbarghazi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Faculty of Advanced Medical Sciences, Department of Applied Cell Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
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18
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Nakamae S, Toba Y, Takayama K, Sakurai F, Mizuguchi H. Nanaomycin A Treatment Promotes Hepatoblast Differentiation from Human iPS Cells. Stem Cells Dev 2018; 27:405-414. [PMID: 29378471 DOI: 10.1089/scd.2017.0251] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research, including drug screening. However, the hepatocyte functions of the HLCs are still lower than those of human hepatocytes. Therefore, we attempted to improve the hepatocyte differentiation method by modulating the DNA epigenetic status. We first examined the expression profiles of the maintenance DNA methyltransferase (DNMT) 1 and the de novo DNMTs DNMT3A and DNMT3B, all of which are essential for mammalian development. Among these DNMTs, the expression levels of DNMT3B were significantly decreased during the hepatoblast differentiation. To accelerate the hepatoblast differentiation, a DNMT3B-selective inhibitor, nanaomycin A, was treated during the hepatoblast differentiation. The gene expression levels of hepatoblast markers (such as alpha-fetoprotein and hepatocyte nuclear factor 4 alpha) were increased by the nanaomycin A treatment. On the other hand, the gene expression levels of hepatoblast markers were decreased by DNMT3B overexpression. These results suggest that it might be possible to promote the hepatoblast differentiation by DNMT3B inhibition using nanaomycin A. Importantly, we also confirmed that the hepatocyte differentiation potency of nanaomycin A-treated hepatoblast-like cells was higher than that of dimethyl sulfoxide-treated hepatoblast-like cells. Our findings should assist in the future generation of functional HLCs for pharmaceutical research.
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Affiliation(s)
- Souichiro Nakamae
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan
| | - Yukiko Toba
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan
| | - Kazuo Takayama
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan .,2 PRESTO, Japan Science and Technology Agency , Saitama, Japan .,3 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation , Health and Nutrition, Osaka, Japan
| | - Fuminori Sakurai
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan
| | - Hiroyuki Mizuguchi
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan .,3 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation , Health and Nutrition, Osaka, Japan .,4 Global Center for Medical Engineering and Informatics, Osaka University , Osaka, Japan
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Guo X, Bai Y, Zhang L, Zhang B, Zagidullin N, Carvalho K, Du Z, Cai B. Cardiomyocyte differentiation of mesenchymal stem cells from bone marrow: new regulators and its implications. Stem Cell Res Ther 2018; 9:44. [PMID: 29482607 PMCID: PMC5828435 DOI: 10.1186/s13287-018-0773-9] [Citation(s) in RCA: 77] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
In the past years, cardiac mortality has decreased, but cardiac diseases are still responsible for millions of deaths every year worldwide. Bone-marrow mesenchymal stem cells (BMSCs) transplantation may be a promising therapeutic strategy because of its capacity to differentiate into cardiac cells. Current research indicates that chemical substances, microRNAs, and cytokines have biological functions that regulate the cardiomyocytes differentiation of BMSCs. In this review, we chiefly summarize the regulatory factors that induce BMSCs to differentiate into cardiomyocytes.
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Affiliation(s)
- Xiaofei Guo
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China
| | - Yan Bai
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China
| | - Li Zhang
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China
| | - Bo Zhang
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China
| | - Naufal Zagidullin
- Department of Internal Diseases, Bashkir State Medical University, Ufa, Russia
| | - Katherine Carvalho
- Cell Therapy and Biotechnology in Regenerative Medicine Research Group, Pequeno Príncipe Faculty, Pelé Pequeno Príncipe Institute, Curitiba, Brazil
| | - Zhimin Du
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China
| | - Benzhi Cai
- Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, Heilongjiang Province, 150081, People's Republic of China.
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20
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Ledford BT, Simmons J, Chen M, Fan H, Barron C, Liu Z, Van Dyke M, He JQ. Keratose Hydrogels Promote Vascular Smooth Muscle Differentiation from C-kit-Positive Human Cardiac Stem Cells. Stem Cells Dev 2017; 26:888-900. [DOI: 10.1089/scd.2016.0351] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Affiliation(s)
- Benjamin T. Ledford
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Jamelle Simmons
- Department of Biomedical Engineering and Mechanics, School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, Virginia
| | - Miao Chen
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Huimin Fan
- Research Institute of Heart Failure, Shanghai East Hospital of Tongji University, Shanghai, People's Republic of China
| | - Catherine Barron
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Zhongmin Liu
- Research Institute of Heart Failure, Shanghai East Hospital of Tongji University, Shanghai, People's Republic of China
| | - Mark Van Dyke
- Department of Biomedical Engineering and Mechanics, School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, Virginia
| | - Jia-Qiang He
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
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21
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Xu X, Wang W, Li Z, Kratz K, Ma N, Lendlein A. Surface geometry of poly(ether imide) boosts mouse pluripotent stem cell spontaneous cardiomyogenesis via modulating the embryoid body formation process. Clin Hemorheol Microcirc 2017; 64:367-382. [DOI: 10.3233/ch-168107] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Affiliation(s)
- Xun Xu
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Weiwei Wang
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
| | - Zhengdong Li
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Karl Kratz
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
| | - Nan Ma
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
| | - Andreas Lendlein
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
- Helmholtz Virtual Institute - Multifunctional Materials in Medicine, Berlin and Teltow, Teltow, Germany
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22
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Wu X, Song M, Yang X, Liu X, Liu K, Jiao C, Wang J, Bai C, Su G, Liu X, Li G. Establishment of bovine embryonic stem cells after knockdown of CDX2. Sci Rep 2016; 6:28343. [PMID: 27320776 PMCID: PMC4913270 DOI: 10.1038/srep28343] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Accepted: 05/31/2016] [Indexed: 01/12/2023] Open
Abstract
Bovine embryonic stem cells (bESCs) have not been successfully established yet. One reason could be that CDX2, as the trophectoderm regulator, expresses in bovine inner cell mass (ICM), which probably becomes a technical barrier for maintaining the pluripotency of bESCs in vitro. We hypothesized that CDX2 knockdown (CDX2-KD) could remove such negative effort, which will be helpful for capturing complete and permanent capacity of pluripotency. Expression and localization of pluripotent genes were not affected in CDX2-KD blastocysts. The CDX2-KD bESCs grew into monolayers on feeder layer. Pluripotent genes expressed at an improved levels and lasted longer time in CDX2-KD bESCs, along with down-regulation of DNA methylation on promoters of both OCT4 and SOX2. The cystic structure typical for trophoblast cells did not show during culturing CDX2-KD bESCs. CDX2-KD bESC-derived Embryoid bodies showed with compact morphology and with the improved levels of differentiations in three germ layers. CDX2-KD bESCs still carried the capacity of forming teratomas with three germ layers after long-term culture. In summary, CDX2 in bovine ICM was inducer of trophoblast lineage with negative effect on maintenance of pluripotency of bESCs. Precise regulation CDX2 expression to switch on/off will be studied next for application on establishment of bESCs.
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Affiliation(s)
- Xia Wu
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Miao Song
- Department of Basic Medicine, Bao Tou Medical College, Bao Tou 014040, China.,Department of Pharmacy, Bao Tou Medical College, Bao Tou 014040, China
| | - Xi Yang
- College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010110, China
| | - Xin Liu
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Kun Liu
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Cuihua Jiao
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Jinze Wang
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Chunling Bai
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Guanghua Su
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Xuefei Liu
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
| | - Guangpeng Li
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010070, China
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23
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Dzobo K, Vogelsang M, Parker MI. Wnt/β-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells. Stem Cell Rev Rep 2016; 11:761-73. [PMID: 26022506 DOI: 10.1007/s12015-015-9598-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Human embryonic stem cells (hESCs) have the potential to differentiate into all cells of the three germ layers, thus making them an attractive source of cells for use in regenerative medicine. The greatest challenge lies in regulating the differentiation of hESCs into specific cell lineages by both intrinsic and extrinsic factors. In this study we determined the effect of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that growth of hESCs on fd-ECM results in hESCs losing their stemness and proliferation potential. As the stem cells differentiate they attain gene expression profiles similar to the primitive streak of the in vivo embryo. The activation of both the MEK-ERK and Wnt/β-catenin signaling pathways is required for the fd-ECM-mediated differentiation of hESCs towards the endoderm and involves integrins α1, α2, α3 and β1. This study illustrates the importance of the cellular microenvironment in directing stem cell fate and that the nature and composition of the extracellular matrix is a crucial determining factor.
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Affiliation(s)
- Kevin Dzobo
- Cape Town Component, Wernher and Beit Building (South), UCT Campus, International Centre for Genetic Engineering and Biotechnology (ICGEB), Anzio Road, Observatory, 7925, Cape Town, South Africa
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24
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Cheng J, Ding Q, Wang J, Deng L, Yang L, Tao L, Lei H, Lu S. 5-Azacytidine delivered by mesoporous silica nanoparticles regulates the differentiation of P19 cells into cardiomyocytes. NANOSCALE 2016; 8:2011-2021. [PMID: 26699243 DOI: 10.1039/c5nr08560h] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/05/2023]
Abstract
Heart disease is one of the deadliest diseases causing mortality due to the limited regenerative capability of highly differentiated cardiomyocytes. Stem cell-based therapy in tissue engineering is one of the most exciting and rapidly growing areas and raises promising prospects for cardiac repair. In this study, we have synthesized FITC-mesoporous silica nanoparticles (FMSNs) based on a sol-gel method (known as Stöber's method) as a drug delivery platform to transport 5-azacytidine in P19 embryonic carcinoma stem cells. The surfactant CTAB is utilized as a liquid crystal template to self-aggregate into micelles, resulting in the synthesis of MSNs. Based on the cell viability assay, treatment with FMSNs + 5-azacytidine resulted in much more significant inhibition of the proliferation than 5-azacytidine alone. To study the mechanism, we have tested the differentiation genes and cardiac marker genes in P19 cells and found that these genes have been up-regulated in P19 embryonic carcinoma stem cells treated with FMSNs + 5-azacytidine + poly(allylamine hydrochloride) (PAH), with the changes of histone modifications on the regulatory region. In conclusion, with FMSNs as drug delivery platforms, 5-azacytidine can be more efficiently delivered into stem cells and can be used to monitor and track the transfection process in situ to clarify their effects on stem cell functions and the differentiation process, which can serve as a promising tool in tissue engineering and other biomedical fields.
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Affiliation(s)
- Jin Cheng
- Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
| | - Qian Ding
- Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China
| | - Jia Wang
- Department of Ultrasound Diagnostics, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China
| | - Lin Deng
- Department of Oncology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China
| | - Lu Yang
- Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China
| | - Lei Tao
- Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China
| | - Haihong Lei
- Department of Neonatology, West China Second University Hospital, Sichuan University, Chengdu 610041, China
| | - Shaoping Lu
- Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
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25
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Cardiotrophin-1 promotes cardiomyocyte differentiation from mouse induced pluripotent stem cells via JAK2/STAT3/Pim-1 signaling pathway. JOURNAL OF GERIATRIC CARDIOLOGY : JGC 2016; 12:591-9. [PMID: 26788034 PMCID: PMC4712363 DOI: 10.11909/j.issn.1671-5411.2015.06.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
BACKGROUND The induced pluripotent stem cell (iPSC) has shown great potential in cellular therapy of myocardial infarction (MI), while its application is hampered by the low efficiency of cardiomyocyte differentiation. The present study was designed to investigate the effects of cardiotrophin-1 (CT-1) on cardiomyocyte differentiation from mouse induced pluripotent stem cells (miPSCs) and the underlying mechanisms involved. METHODS The optimal treatment condition for cardiomyocyte differentiation from miPSCs was established with ideal concentration (10 ng/mL) and duration (from day 3 to day 14) of CT-1 administration. Up-regulated expression of cardiac specific genes that accounted for embryonic cardiogenesis was observed by quantitative RT-PCR. Elevated amount of α-myosin heavy chain (α-MHC) and cardiac troponin I (cTn I) positive cells were detected by immunofluorescence staining and flow cytometry analysis in CT-1 group. RESULTS Transmission electron microscopic analysis revealed that cells treated with CT-1 showed better organized sacromeric structure and more mitochondria, which are morphological characteristic of matured cardiomyocytes. Western blot demonstrated that CT-1 promotes cardiomyocyte differentiation from miPSCs partly via JAK2/STAT3/Pim-1 pathway as compared with control group. CONCLUSIONS These findings suggested that CT-1 could enhance the cardiomyocyte differentiation as well as the maturation of mouse induced pluripotent stem cell derived cardiomyocytes by regulating JAK2/STAT3/Pim-1signaling pathway.
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Abstract
The advent of pluripotent stem cells (PSCs) enabled a multitude of studies for modeling the development of diseases and testing pharmaceutical therapeutic potential in vitro. These PSCs have been differentiated to multiple cell types to demonstrate its pluripotent potential, including cardiomyocytes (CMs). However, the efficiency and efficacy of differentiation vary greatly between different cell lines and methods. Here, we describe two different methods for acquiring CMs from human pluripotent lines. One method involves the generation of embryoid bodies, which emulates the natural developmental process, while the other method chemically activates the canonical Wnt signaling pathway to induce a monolayer of cardiac differentiation.
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Affiliation(s)
| | - Elisa Di Pasquale
- Humanitas Clinical and Research Center, Rozzano, MI, Italy.
- Istituto di Ricerca Genetica e Biomedica, National Research Council of Italy, Milan, Italy.
- C/O Humanitas Clinical and Research Center, Via Manzoni, 56, 20089, Rozzano, MI, Italy.
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27
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Lin HY, Lee DC, Wang HD, Chi YH, Chiu IM. Activation of FGF1B Promoter and FGF1 Are Involved in Cardiogenesis Through the Signaling of PKC, but Not MAPK. Stem Cells Dev 2015; 24:2853-63. [DOI: 10.1089/scd.2015.0157] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Hung-Yu Lin
- Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
- Graduate Program of Biotechnology in Medicine, Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
- Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
| | - Don-Ching Lee
- Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
| | - Horng-Dar Wang
- Graduate Program of Biotechnology in Medicine, Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
- Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
| | - Ya-Hui Chi
- Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
- Graduate Program of Biotechnology in Medicine, Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
| | - Ing-Ming Chiu
- Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
- Graduate Program of Biotechnology in Medicine, Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
- Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
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28
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Human cardiomyocyte generation from pluripotent stem cells: A state-of-art. Life Sci 2015; 145:98-113. [PMID: 26682938 DOI: 10.1016/j.lfs.2015.12.023] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Revised: 11/08/2015] [Accepted: 12/09/2015] [Indexed: 12/11/2022]
Abstract
The human heart is considered a non-regenerative organ. Worldwide, cardiovascular diseases continue to be the leading cause of death. Despite advances in cardiac treatment, myocardial repair remains severely limited by the lack of an appropriate source of viable cardiomyocytes (CMs) to replace damaged tissue. Human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can efficiently be differentiated into functional CMs necessary for cell replacement therapy and other potential applications. The number of protocols that derive CMs from hPSCs has increased exponentially over the past decade following observation of the first human beating CMs. A number of highly efficient, chemical based protocols have been developed to generate human CMs (hCMs) in small-scale and large-scale suspension systems. To reduce the heterogeneity of hPSC-derived CMs, the differentiation protocols were modulated to exclusively generate atrial-, ventricular-, and nodal-like CM subtypes. Recently, remarkable advances have been achieved in hCM generation including chemical-based cardiac differentiation, cardiac subtype specification, large-scale suspension culture differentiation, and development of chemically defined culture conditions. These hCMs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues are overcome. Herein we review recent progress in the in vitro generation of CMs and cardiac subtypes from hPSCs and discuss their potential applications and current limitations.
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29
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Park D, Lim J, Park JY, Lee SH. Concise Review: Stem Cell Microenvironment on a Chip: Current Technologies for Tissue Engineering and Stem Cell Biology. Stem Cells Transl Med 2015; 4:1352-68. [PMID: 26450425 DOI: 10.5966/sctm.2015-0095] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 07/29/2015] [Indexed: 01/09/2023] Open
Abstract
UNLABELLED Stem cells have huge potential in many therapeutic areas. With conventional cell culture methods, however, it is difficult to achieve in vivo-like microenvironments in which a number of well-controlled stimuli are provided for growing highly sensitive stem cells. In contrast, microtechnology-based platforms offer advantages of high precision, controllability, scalability, and reproducibility, enabling imitation of the complex physiological context of in vivo. This capability may fill the gap between the present knowledge about stem cells and that required for clinical stem cell-based therapies. We reviewed the various types of microplatforms on which stem cell microenvironments are mimicked. We have assigned the various microplatforms to four categories based on their practical uses to assist stem cell biologists in using them for research. In particular, many examples are given of microplatforms used for the production of embryoid bodies and aggregates of stem cells in vitro. We also categorized microplatforms based on the types of factors controlling the behaviors of stem cells. Finally, we outline possible future directions for microplatform-based stem cell research, such as research leading to the production of well-defined environments for stem cells to be used in scaled-up systems or organs-on-a-chip, the regulation of induced pluripotent stem cells, and the study of the genetic states of stem cells on microplatforms. SIGNIFICANCE Stem cells are highly sensitive to a variety of physicochemical cues, and their fate can be easily altered by a slight change of environment; therefore, systematic analysis and discrimination of the extracellular signals and intracellular pathways controlling the fate of cells and experimental realization of sensitive and controllable niche environments are critical. This review introduces diverse microplatforms to provide in vitro stem cell niches. Microplatforms could control microenvironments around cells and have recently attracted much attention in biology including stem cell research. These microplatforms and the future directions of stem cell microenvironment are described.
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Affiliation(s)
- DoYeun Park
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea
| | - Jaeho Lim
- School of Biomedical Engineering, College of Health Science, Korea University, Seoul, Republic of Korea
| | - Joong Yull Park
- School of Mechanical Engineering, College of Engineering, Chung-ang University, Seoul, Republic of Korea
| | - Sang-Hoon Lee
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea School of Biomedical Engineering, College of Health Science, Korea University, Seoul, Republic of Korea
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30
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Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro. Stem Cells Int 2015; 2015:247091. [PMID: 26357517 PMCID: PMC4556334 DOI: 10.1155/2015/247091] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2015] [Revised: 06/27/2015] [Accepted: 07/02/2015] [Indexed: 12/04/2022] Open
Abstract
Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.
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31
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Szebényi K, Péntek A, Erdei Z, Várady G, Orbán TI, Sarkadi B, Apáti Á. Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression. Tissue Eng Part C Methods 2015; 21:35-45. [PMID: 24734786 DOI: 10.1089/ten.tec.2013.0646] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFP(high) rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFP(high) rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFP(high) rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications.
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Affiliation(s)
- Kornélia Szebényi
- 1 Institute of Molecular Pharmacology , Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
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32
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Talkhabi M, Pahlavan S, Aghdami N, Baharvand H. Ascorbic acid promotes the direct conversion of mouse fibroblasts into beating cardiomyocytes. Biochem Biophys Res Commun 2015; 463:699-705. [PMID: 26047705 DOI: 10.1016/j.bbrc.2015.05.127] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2015] [Accepted: 05/31/2015] [Indexed: 12/14/2022]
Abstract
Recent advances in the direct conversion of fibroblasts to cardiomyocytes suggest this process as a novel promising approach for cardiac cell-based therapies. Here, by screening the effects of 10 candidate small molecules along with transient overexpression of Yamanaka factors, we show ascorbic acid (AA), also known as vitamin C, enhances reprogramming of mouse fibroblasts into beating cardiomyocytes. Immunostaining and gene expression analyses for pluripotency and cardiac lineage markers confirmed beating patches were derived from non-cardiac lineage cells without passing through a pluripotent intermediate. Further analysis revealed that AA also increased the size of the beating areas and the number of cardiac progenitors. Immunostaining for cardiac markers, as well as electrophysiological analysis confirmed the functionality of directly converted cardiomyocytes. These results illustrate the importance of AA in direct conversion of fibroblasts to cardiomyocytes and may open new insights into future biomedical applications for induced cardiomyocytes.
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Affiliation(s)
- Mahmood Talkhabi
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
| | - Sara Pahlavan
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran.
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Wu YJ, Chen SY, Chang SJ, Kuo SM. Enhanced differentiation of rat MSCs into cardiomyocytes with 5-azacytidine/collagen I nano-molecules. ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. ANNUAL INTERNATIONAL CONFERENCE 2015; 2013:322-5. [PMID: 24109689 DOI: 10.1109/embc.2013.6609502] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
This study was to investigate the enhancement ability of 5-azacytidine (5-aza) and collagen I nano-molecules treatment to the differentiation of rat mesenchymal stem cells (MSCs) towards a cardiomyocytes in vitro. The results demonstrated that the size of the cells increased significantly and connecting with adjoining cells by forming myotube-like structures. Also, additional treatment of the MSCs with collagen I nano-fibrils significantly increased two transcription factors GATA-4 and Nkx2.5 expressions and three expressions of cardiac genes of troponin I, β-myosin heavy chain and cardiac α-actin compared with MSC groups treated only with 5-aza at early 3 d culturing(all, P<0.01 or better). These results indicate that culturing MSCs with collagen I nano-molecules, which could act as scaffolds or soluble protein ingredients, leads to alterations in gene expression and affects the differentiation fate induced with 5-aza.
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Pettinato G, Wen X, Zhang N. Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells. Stem Cells Dev 2015; 24:1595-609. [PMID: 25900308 DOI: 10.1089/scd.2014.0427] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell-cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed.
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Affiliation(s)
- Giuseppe Pettinato
- 1 Department of Biomedical Engineering, Virginia Commonwealth University , Richmond, Virginia.,2 Department of Chemical and Life Science Engineering, Virginia Commonwealth University , Richmond, Virginia
| | - Xuejun Wen
- 2 Department of Chemical and Life Science Engineering, Virginia Commonwealth University , Richmond, Virginia.,3 Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, Shanghai, People's Republic of China
| | - Ning Zhang
- 1 Department of Biomedical Engineering, Virginia Commonwealth University , Richmond, Virginia
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Zhao S, Agarwal P, Rao W, Huang H, Zhang R, Liu Z, Yu J, Weisleder N, Zhang W, He X. Coaxial electrospray of liquid core-hydrogel shell microcapsules for encapsulation and miniaturized 3D culture of pluripotent stem cells. Integr Biol (Camb) 2015; 6:874-84. [PMID: 25036382 DOI: 10.1039/c4ib00100a] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on the 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic.
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Affiliation(s)
- Shuting Zhao
- Department of Biomedical Engineering, The Ohio State University, 1080 Carmack Road, Columbus, OH 43210, USA.
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Shabani P, Ghazizadeh Z, Pahlavan S, Hashemizadeh S, Baharvand H, Aghdami N, Doosti M. Exogenous treatment with eicosapentaenoic acid supports maturation of cardiomyocytes derived from embryonic stem cells. Biochem Biophys Res Commun 2015; 461:281-6. [PMID: 25871791 DOI: 10.1016/j.bbrc.2015.04.018] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2015] [Accepted: 04/03/2015] [Indexed: 11/29/2022]
Abstract
Embryonic stem cells offer multiple advantages over adult stem cells in terms of achieving acceptable number of functional cardiomyocytes to be exploited in cell therapy. However, differentiation efficacy is still a major issue to be solved before moving to regenerative medicine. Although a vast number of chemical compounds have been tested on efficiency of cardiac differentiation, the effect of fish oil components, such as eicosapentaenoic acid (EPA) on developmental bioenergetics, and hence cardiac differentiation, remained unstudied. EPA has been reported to have several cardioprotective effects, but there is no study addressing its role in cardiac differentiation. After mesoderm induction of embryoid bodies (EBs) derived from mouse embryonic stem cells (mESCs) in hanging drops initiated by ascorbic acid, they were treated with various concentrations of EPA. Gene and protein expression and functional properties of cardiomyocytes derived from ESCs were evaluated following treatment with various concentrations of EPA. Exposure to low concentrations of EPA (10 μM) increased percentage of beating colonies and beating area. This treatment also resulted in up to 3 fold increase in expression of NKX2-5, MEF2C, MYH6, TNNT2 and CX43. FACS analysis confirmed gene expression analysis with increased percentage of MYH6 positive cells in EPA-treated group compared to the control group. In contrast, the expression of genes coding for cardiac differentiation, remained constant or even declined with higher concentrations of EPA. In conclusion, we have demonstrated that treatment of mESCs undergoing cardiac differentiation with low concentration, but not high concentration of EPA up-regulate transcription of genes associated with cardiac development.
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Affiliation(s)
- Parisa Shabani
- Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran; Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Zaniar Ghazizadeh
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sara Pahlavan
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Shiva Hashemizadeh
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - Mahmood Doosti
- Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
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Liu KC, Yo YT, Huang RL, Wang YC, Liao YP, Huang TS, Chao TK, Lin CK, Weng SJ, Ma KH, Chang CC, Yu MH, Lai HC. Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor. Oncotarget 2014; 4:2366-82. [PMID: 24280306 PMCID: PMC3926833 DOI: 10.18632/oncotarget.1424] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Spheroid formation is one property of stem cells—such as embryo-derived or neural stem cells—that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial–mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers. This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer.
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Affiliation(s)
- Kuei-Chun Liu
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
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Pettinato G, Vanden Berg-Foels WS, Zhang N, Wen X. ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells. PLoS One 2014; 9:e100742. [PMID: 25365581 PMCID: PMC4217711 DOI: 10.1371/journal.pone.0100742] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2014] [Accepted: 05/30/2014] [Indexed: 12/18/2022] Open
Abstract
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.
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Affiliation(s)
- Giuseppe Pettinato
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
| | - Wendy S. Vanden Berg-Foels
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America
| | - Ning Zhang
- Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- * E-mail: (NZ); (XW)
| | - Xuejun Wen
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America
- Institute for Biomedical Engineering and Nano Science (iNANO), Shanghai East Hospital, Tongji Medical School, Tongji University, Shanghai, People's Republic of China
- * E-mail: (NZ); (XW)
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A microwell pattern for C17.2 cell aggregate formation with concave cylindrical surface induced cell peeling. Biomaterials 2014; 35:9423-37. [DOI: 10.1016/j.biomaterials.2014.07.046] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2014] [Accepted: 07/23/2014] [Indexed: 01/17/2023]
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Atkinson SP, Lako M, Armstrong L. Potential for pharmacological manipulation of human embryonic stem cells. Br J Pharmacol 2014; 169:269-89. [PMID: 22515554 DOI: 10.1111/j.1476-5381.2012.01978.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products that can be cost-prohibitive for large-scale production, and effective lineage-specific differentiation protocols also still remain relatively undefined and are inefficient at producing large amounts of cells for therapeutic use. Furthermore, the mechanisms and signalling pathways that mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large-scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents that can boost hESC pluripotency/self-renewal and survival and has greatly increased the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future.
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41
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Lam ATL, Chen AKL, Li J, Birch WR, Reuveny S, Oh SKW. Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture. Stem Cell Res Ther 2014; 5:110. [PMID: 25223792 PMCID: PMC4183116 DOI: 10.1186/scrt498] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2014] [Accepted: 09/09/2014] [Indexed: 12/27/2022] Open
Abstract
Introduction Myocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed. Methods Production of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process. Results In the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316 ± 11 μm. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9 × 106 CM/ml, as compared to 0.5 × 106 CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3 × 106 CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 μM) prolonged the QT-interval duration by 40% and verapamil (3 μM) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays. Conclusions We have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies. Electronic supplementary material The online version of this article (doi:10.1186/scrt498) contains supplementary material, which is available to authorized users.
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42
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Han Y, Bai T, Liu W. Controlled heterogeneous stem cell differentiation on a shape memory hydrogel surface. Sci Rep 2014; 4:5815. [PMID: 25068211 PMCID: PMC5376171 DOI: 10.1038/srep05815] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2014] [Accepted: 07/07/2014] [Indexed: 12/29/2022] Open
Abstract
The success of stem cell therapies is highly dependent on the ability to control their programmed differentiation. So far, it is commonly believed that the differentiation behavior of stem cells is supposed to be identical when they are cultured on the same homogeneous platform. However, in this report, we show that this is not always true. By utilizing a double-ion-triggered shape memory effect, the pre-seeded hMSCs were controllably located in different growth positions. Here, we demonstrate for the first time that the differentiation behavior of hMSCs is highly sensitive to their growth position on a hydrogel scaffold. This work will not only enrich the mechanisms for controlling the differentiation of stem cells, but also offer a one-of-a-kind platform to achieve a heterogeneously differentiated stem cell-seeded hydrogel scaffold for complex biological applications.
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Affiliation(s)
- Yanjiao Han
- 1] Collaborative Innovation Center of Chemical Science and Engineering(Tianjin), Tianjin 300072, P. R. China [2]
| | - Tao Bai
- 1] Collaborative Innovation Center of Chemical Science and Engineering(Tianjin), Tianjin 300072, P. R. China [2]
| | - Wenguang Liu
- Collaborative Innovation Center of Chemical Science and Engineering(Tianjin), Tianjin 300072, P. R. China
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Zuellig RA, Cavallari G, Gerber P, Tschopp O, Spinas GA, Moritz W, Lehmann R. Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets. J Tissue Eng Regen Med 2014; 11:109-120. [PMID: 24737702 DOI: 10.1002/term.1891] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2013] [Revised: 12/20/2013] [Accepted: 02/26/2014] [Indexed: 01/01/2023]
Abstract
Previously we demonstrated the superiority of small islets vs large islets in terms of function and survival after transplantation, and we generated reaggregated rat islets (pseudo-islets) of standardized small dimensions by the hanging-drop culture method (HDCM). The aim of this study was to generate human pseudo-islets by HDCM and to evaluate and compare the physiological properties of rat and human pseudo-islets. Isolated rat and human islets were dissociated into single cells and incubated for 6-14 days by HDCM. Newly formed pseudo-islets were analysed for dimensions, morphology, glucose-stimulated insulin secretion (GSIS) and total insulin content. The morphology of reaggregated human islets was similar to that of native islets, while rat pseudo-islets had a reduced content of α and δ cells. GSIS of small rat and human pseudo-islets (250 cells) was increased up to 4.0-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, when compared to their native counterparts. Human pseudo-islets showed a more pronounced first-phase insulin secretion as compared to intact islets. GSIS was inversely correlated to islet size, and small islets (250 cells) contained up to six-fold more insulin/cell than large islets (1500 cells). Tissue loss with this new technology could be reduced to 49.2 ± 1.5% in rat islets, as compared to the starting amount. With HDCM, pseudo-islets of standardized size with similar cellular composition and improved biological function can be generated, which compensates for tissue loss during production. Transplantation of small pseudo-islets may represent an attractive strategy to improve graft survival and function, due to better oxygen and nutrient supply during the phase of revascularization. Copyright © 2014 John Wiley & Sons, Ltd.
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Affiliation(s)
- R A Zuellig
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - G Cavallari
- Nephrology, Dialysis and Transplantation Unit (Stefoni), S.Orsola-Malpighi Hospital, University of Bologna, Italy
| | - P Gerber
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - O Tschopp
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - G A Spinas
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - W Moritz
- InSphero AG, Schlieren, Switzerland
| | - R Lehmann
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
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Kinney MA, Hookway TA, Wang Y, McDevitt TC. Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics. Ann Biomed Eng 2014; 42:352-67. [PMID: 24297495 PMCID: PMC3939035 DOI: 10.1007/s10439-013-0953-9] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2013] [Accepted: 11/21/2013] [Indexed: 12/11/2022]
Abstract
The physiochemical stem cell microenvironment regulates the delicate balance between self-renewal and differentiation. The three-dimensional assembly of stem cells facilitates cellular interactions that promote morphogenesis, analogous to the multicellular, heterotypic tissue organization that accompanies embryogenesis. Therefore, expansion and differentiation of stem cells as multicellular aggregates provides a controlled platform for studying the biological and engineering principles underlying spatiotemporal morphogenesis and tissue patterning. Moreover, three-dimensional stem cell cultures are amenable to translational screening applications and therapies, which underscores the broad utility of scalable suspension cultures across laboratory and clinical scales. In this review, we discuss stem cell morphogenesis in the context of fundamental biophysical principles, including the three-dimensional modulation of adhesions, mechanics, and molecular transport and highlight the opportunities to employ stem cell spheroids for tissue modeling, bioprocessing, and regenerative therapies.
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Affiliation(s)
- Melissa A. Kinney
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University, Atlanta, GA, USA
| | - Tracy A. Hookway
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University, Atlanta, GA, USA
| | - Yun Wang
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University, Atlanta, GA, USA
| | - Todd C. McDevitt
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University, Atlanta, GA, USA
- The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA
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Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/Akt pathways. Int J Mol Sci 2014; 15:605-28. [PMID: 24398984 PMCID: PMC3907828 DOI: 10.3390/ijms15010605] [Citation(s) in RCA: 127] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2013] [Revised: 12/21/2013] [Accepted: 01/02/2014] [Indexed: 12/26/2022] Open
Abstract
In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-β1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-β/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-β/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-β/SMAD2 and PI3K/AKT pathways.
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Olmer R, Martin U. Induced Pluripotent Stem Cells Differentiate into Functional Cardiomyocytes. STEM CELLS AND CANCER STEM CELLS, VOLUME 12 2014. [DOI: 10.1007/978-94-017-8032-2_5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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47
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Engineering Cellular Assembly for Applications in Regenerative Medicine. NANOMEDICINE AND NANOTOXICOLOGY 2014. [DOI: 10.1007/978-4-431-55139-3_6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Lin PY, Hung SH, Yang YC, Liao LC, Hsieh YC, Yen HJ, Lu HE, Lee MS, Chu IM, Hwang SM. A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells. Stem Cells Dev 2013; 23:372-9. [PMID: 24083371 DOI: 10.1089/scd.2013.0253] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.
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Affiliation(s)
- Pei-Yi Lin
- 1 Bioresource Collection and Research Center, Food Industry Research and Development Institute , Hsinchu, Taiwan
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Prabhakaran MP, Mobarakeh LG, Kai D, Karbalaie K, Nasr-Esfahani MH, Ramakrishna S. Differentiation of embryonic stem cells to cardiomyocytes on electrospun nanofibrous substrates. J Biomed Mater Res B Appl Biomater 2013; 102:447-54. [PMID: 24039141 DOI: 10.1002/jbm.b.33022] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2012] [Revised: 06/24/2013] [Accepted: 08/01/2013] [Indexed: 12/31/2022]
Abstract
The potential of pluripotent embryonic stem cells (ESCs) isolated from the inner mass of blastocysts are investigated for its ability to differentiate on biocompatible electrospun nanofibers, for regeneration of the myocardially infracted heart. Nanostructured poly(d,l-lactide-co-glycolide)/collagen (PLGA/Col) scaffolds with fiber diameters in the range of 300 ± 65 nm, was fabricated by electrospinning to mimic the extracellular matrix of the native tissue. During the culture of embryoid bodies outgrowth on the scaffolds, and further differentiation of ESCs to cardiomyocytes, the PLGA/Col nanofibers was found better than that of the electrospun PLGA nanofibers, where a better interaction and growth of ESC differentiated cardiomyocytes was observed on the composite scaffolds. The phenotypical characteristics of ESC-derived cardiomyocytes and molecular protein expression were carried out by scanning electron microscopy and immunocytochemistry, respectively. Our studies highlight the significance of a suitable material, its architecture, and cell-biomaterial interactions that is essential at a nanoscale level signifying the application of a bioengineered cardiac graft for stem cell differentiation and transplantation, which could be an intriguing strategy for cardiac regeneration.
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Affiliation(s)
- Molamma P Prabhakaran
- Center for Nanofibers and Nanotechnology, E3-05-14, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, Singapore, 117576
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Chow MZ, Boheler KR, Li RA. Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives. Stem Cell Res Ther 2013; 4:97. [PMID: 23953772 PMCID: PMC3854712 DOI: 10.1186/scrt308] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Heart diseases remain a major cause of mortality and morbidity worldwide. However, terminally differentiated human adult cardiomyocytes (CMs) possess a very limited innate ability to regenerate. Directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into CMs has enabled clinicians and researchers to pursue the novel therapeutic paradigm of cell-based cardiac regeneration. In addition to tissue engineering and transplantation studies, the need for functional CMs has also prompted researchers to explore molecular pathways and develop strategies to improve the quality, purity and quantity of hESC-derived and iPSC-derived CMs. In this review, we describe various approaches in directed CM differentiation and driven maturation, and discuss potential limitations associated with hESCs and iPSCs, with an emphasis on the role of epigenetic regulation and chromatin remodeling, in the context of the potential and challenges of using hESC-CMs and iPSC-CMs for drug discovery and toxicity screening, disease modeling, and clinical applications.
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Affiliation(s)
- Maggie Zi Chow
- Stem Cell and Regenerative Medicine Consortium, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong Jockey Club Building for Interdisciplinary Research, Pokfulam, Hong Kong
- Department of Physiology, The University of Hong Kong, 4th Floor, 21 Sassoon Road, Laboratory Block, Faculty of Medicine Building, Pokfulam, Hong Kong
| | - Kenneth R Boheler
- Stem Cell and Regenerative Medicine Consortium, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong Jockey Club Building for Interdisciplinary Research, Pokfulam, Hong Kong
- Department of Physiology, The University of Hong Kong, 4th Floor, 21 Sassoon Road, Laboratory Block, Faculty of Medicine Building, Pokfulam, Hong Kong
- Molecular Cardiology and Stem Cell Unit, Laboratory of Cardiovascular Sciences, National Institute on Aging, National Institutes of Health, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA
| | - Ronald A Li
- Stem Cell and Regenerative Medicine Consortium, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong Jockey Club Building for Interdisciplinary Research, Pokfulam, Hong Kong
- Department of Physiology, The University of Hong Kong, 4th Floor, 21 Sassoon Road, Laboratory Block, Faculty of Medicine Building, Pokfulam, Hong Kong
- Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1234, New York, New York 10029-6574, USA
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