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Yoshida T, Tsukamoto M, Kimura K, Tanaka M, Kuwamura M, Hatoya S. Establishment of feline embryonic stem cells from the inner cell mass of blastocysts produced in vitro. Regen Ther 2025; 28:63-72. [PMID: 39697661 PMCID: PMC11652941 DOI: 10.1016/j.reth.2024.11.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Revised: 10/12/2024] [Accepted: 11/17/2024] [Indexed: 12/20/2024] Open
Abstract
Introduction The rising number of cats as pets and the growing interest in animal welfare have led to an increased need for the latest treatments in feline veterinary medicine. Among these, veterinary regenerative medicine using pluripotent stem cells is gaining significant attention. However, there have been no reports on establishing feline embryonic stem cell (ESC) lines that possess the pluripotent potential and the ability to differentiate into three germ layers. Methods In this study, we isolated three inner cell masses from feline in vitro-derived blastocysts and subcultured them in a chemically defined medium (StemFit AK02N). We assessed the expression of undifferentiated markers, the ability to differentiate into the three germ layers, and the karyotype structure. Results We established three feline ESC lines. Feline ESCs exhibited positive staining for alkaline phosphatase. RT-qPCR analysis revealed that these cells express undifferentiated marker genes in vitro. Immunostaining and flow cytometry analysis demonstrated that feline ESCs express undifferentiated marker proteins in vitro. In the KSR/FBS medium with or without Activin A, feline ESCs differentiated into all three germ layers (ectoderm, endoderm, and mesoderm), expressing specific marker genes and proteins for each germ layer, as evidenced by RT-qPCR, immunostaining, and flow cytometry. Furthermore, we confirmed that feline ESCs formed teratomas comprising all three germ layers in mouse testes, demonstrating de novo pluripotency in vivo. We also verified that the feline ESCs maintained a normal karyotype. Conclusions We successfully established three feline ESC lines, each possessing pluripotent potential and capable of differentiating into all three germ layers, derived from the inner cell masses of blastocysts produced in vitro.
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Affiliation(s)
- Takumi Yoshida
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Masaya Tsukamoto
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Kazuto Kimura
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Miyuu Tanaka
- Department of Integrated Structural Biosciences, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Mitsuru Kuwamura
- Department of Integrated Structural Biosciences, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
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Nwokoye PN, Abilez OJ. Bioengineering methods for vascularizing organoids. CELL REPORTS METHODS 2024; 4:100779. [PMID: 38759654 PMCID: PMC11228284 DOI: 10.1016/j.crmeth.2024.100779] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Revised: 03/01/2024] [Accepted: 04/24/2024] [Indexed: 05/19/2024]
Abstract
Organoids, self-organizing three-dimensional (3D) structures derived from stem cells, offer unique advantages for studying organ development, modeling diseases, and screening potential therapeutics. However, their translational potential and ability to mimic complex in vivo functions are often hindered by the lack of an integrated vascular network. To address this critical limitation, bioengineering strategies are rapidly advancing to enable efficient vascularization of organoids. These methods encompass co-culturing organoids with various vascular cell types, co-culturing lineage-specific organoids with vascular organoids, co-differentiating stem cells into organ-specific and vascular lineages, using organoid-on-a-chip technology to integrate perfusable vasculature within organoids, and using 3D bioprinting to also create perfusable organoids. This review explores the field of organoid vascularization, examining the biological principles that inform bioengineering approaches. Additionally, this review envisions how the converging disciplines of stem cell biology, biomaterials, and advanced fabrication technologies will propel the creation of increasingly sophisticated organoid models, ultimately accelerating biomedical discoveries and innovations.
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Affiliation(s)
- Peter N Nwokoye
- Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Oscar J Abilez
- Department of Cardiothoracic Surgery, Stanford University, Stanford, CA 94305, USA; Division of Pediatric CT Surgery, Stanford University, Stanford, CA 94305, USA; Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA; Maternal and Child Health Research Institute, Stanford University, Stanford, CA 94305, USA; Bio-X Program, Stanford University, Stanford, CA 94305, USA.
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Nwokoye PN, Abilez OJ. Blood vessels in a dish: the evolution, challenges, and potential of vascularized tissues and organoids. Front Cardiovasc Med 2024; 11:1336910. [PMID: 38938652 PMCID: PMC11210405 DOI: 10.3389/fcvm.2024.1336910] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2023] [Accepted: 04/19/2024] [Indexed: 06/29/2024] Open
Abstract
Vascular pathologies are prevalent in a broad spectrum of diseases, necessitating a deeper understanding of vascular biology, particularly in overcoming the oxygen and nutrient diffusion limit in tissue constructs. The evolution of vascularized tissues signifies a convergence of multiple scientific disciplines, encompassing the differentiation of human pluripotent stem cells (hPSCs) into vascular cells, the development of advanced three-dimensional (3D) bioprinting techniques, and the refinement of bioinks. These technologies are instrumental in creating intricate vascular networks essential for tissue viability, especially in thick, complex constructs. This review provides broad perspectives on the past, current state, and advancements in key areas, including the differentiation of hPSCs into specific vascular lineages, the potential and challenges of 3D bioprinting methods, and the role of innovative bioinks mimicking the native extracellular matrix. We also explore the integration of biophysical cues in vascularized tissues in vitro, highlighting their importance in stimulating vessel maturation and functionality. In this review, we aim to synthesize these diverse yet interconnected domains, offering a broad, multidisciplinary perspective on tissue vascularization. Advancements in this field will help address the global organ shortage and transform patient care.
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Affiliation(s)
- Peter N. Nwokoye
- Department of Medicine, Stanford University School of Medicine, Stanford, CA, United States
| | - Oscar J. Abilez
- Department of Cardiothoracic Surgery, Stanford University, Stanford, CA, United States
- Division of Pediatric CT Surgery, Stanford University, Stanford, CA, United States
- Cardiovascular Institute, Stanford University, Stanford, CA, United States
- Maternal and Child Health Research Institute, Stanford University, Stanford, CA, United States
- Bio-X Program, Stanford University, Stanford, CA, United States
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da Silva MDV, Piva M, Martelossi-Cebinelli G, Stinglin Rosa Ribas M, Hoffmann Salles Bianchini B, K Heintz O, Casagrande R, Verri WA. Stem cells and pain. World J Stem Cells 2023; 15:1035-1062. [PMID: 38179216 PMCID: PMC10762525 DOI: 10.4252/wjsc.v15.i12.1035] [Citation(s) in RCA: 2] [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: 07/28/2023] [Revised: 11/06/2023] [Accepted: 11/30/2023] [Indexed: 12/26/2023] Open
Abstract
Pain can be defined as an unpleasant sensory and emotional experience caused by either actual or potential tissue damage or even resemble that unpleasant experience. For years, science has sought to find treatment alternatives, with minimal side effects, to relieve pain. However, the currently available pharmacological options on the market show significant adverse events. Therefore, the search for a safer and highly efficient analgesic treatment has become a priority. Stem cells (SCs) are non-specialized cells with a high capacity for replication, self-renewal, and a wide range of differentiation possibilities. In this review, we provide evidence that the immune and neuromodulatory properties of SCs can be a valuable tool in the search for ideal treatment strategies for different types of pain. With the advantage of multiple administration routes and dosages, therapies based on SCs for pain relief have demonstrated meaningful results with few downsides. Nonetheless, there are still more questions than answers when it comes to the mechanisms and pathways of pain targeted by SCs. Thus, this is an evolving field that merits further investigation towards the development of SC-based analgesic therapies, and this review will approach all of these aspects.
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Affiliation(s)
- Matheus Deroco Veloso da Silva
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, State University of Londrina, Londrina 86057-970, Paraná, Brazil
| | - Maiara Piva
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, State University of Londrina, Londrina 86057-970, Paraná, Brazil
| | - Geovana Martelossi-Cebinelli
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, State University of Londrina, Londrina 86057-970, Paraná, Brazil
| | - Mariana Stinglin Rosa Ribas
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, State University of Londrina, Londrina 86057-970, Paraná, Brazil
| | - Beatriz Hoffmann Salles Bianchini
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, State University of Londrina, Londrina 86057-970, Paraná, Brazil
| | - Olivia K Heintz
- Morningside Graduate School of Biomedical Sciences, University of Massachusetts Chan Medical School, Worcester, MA 01655, United States
| | - Rubia Casagrande
- Department of Pharmaceutical Sciences, Center of Health Science, State University of Londrina, Londrina 86038-440, Paraná, Brazil
| | - Waldiceu A Verri
- Department of Pathology, Laboratory of Pain, Inflammation, Neuropathy and Cancer, Center of Biological Sciences, State University of Londrina, Londrina 86057-970, Paraná, Brazil.
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Fang Q, Wei Y, Zhang Y, Cao W, Yan L, Kong M, Zhu Y, Xu Y, Guo L, Zhang L, Wang W, Yu Y, Sun J, Yang J. Stem cells as potential therapeutics for hearing loss. Front Neurosci 2023; 17:1259889. [PMID: 37746148 PMCID: PMC10512725 DOI: 10.3389/fnins.2023.1259889] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Accepted: 08/23/2023] [Indexed: 09/26/2023] Open
Abstract
Hearing impairment is a global health problem. Stem cell therapy has become a cutting-edge approach to tissue regeneration. In this review, the recent advances in stem cell therapy for hearing loss have been discussed. Nanomaterials can modulate the stem cell microenvironment to augment the therapeutic effects further. The potential of combining nanomaterials with stem cells for repairing and regenerating damaged inner ear hair cells (HCs) and spiral ganglion neurons (SGNs) has also been discussed. Stem cell-derived exosomes can contribute to the repair and regeneration of damaged tissue, and the research progress on exosome-based hearing loss treatment has been summarized as well. Despite stem cell therapy's technical and practical limitations, the findings reported so far are promising and warrant further investigation for eventual clinical translation.
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Affiliation(s)
- Qiaojun Fang
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
- School of Life Sciences and Technology, Southeast University, Nanjing, China
| | - Yongjie Wei
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Yuhua Zhang
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Wei Cao
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Lin Yan
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Mengdie Kong
- School of Life Sciences and Technology, Southeast University, Nanjing, China
| | - Yongjun Zhu
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Yan Xu
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Lingna Guo
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Lei Zhang
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Weiqing Wang
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Yafeng Yu
- Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
| | - Jingwu Sun
- Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Jianming Yang
- Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
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A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells. Genes (Basel) 2022; 13:genes13122363. [PMID: 36553630 PMCID: PMC9777998 DOI: 10.3390/genes13122363] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2022] [Revised: 11/30/2022] [Accepted: 12/03/2022] [Indexed: 12/16/2022] Open
Abstract
Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.
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7
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Agarwal D, Kuhns R, Dimitriou CN, Barlow E, Wahlin KJ, Enke RA. Bulk RNA sequencing analysis of developing human induced pluripotent cell-derived retinal organoids. Sci Data 2022; 9:759. [PMID: 36494376 PMCID: PMC9734101 DOI: 10.1038/s41597-022-01853-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2022] [Accepted: 11/21/2022] [Indexed: 12/13/2022] Open
Abstract
Retinogenesis involves the transformation of the anterior developing brain into organized retinal lamellae coordinated by intricate gene signalling networks. This complex process has been investigated in several model organisms such as birds, fish, mammals and amphibians, yet many facets of retinal development are different in humans and remain unexplored. In this regard, human pluripotent stem cell (hPSC)-derived 3D retinal organoids and Next Generation Sequencing (NGS) have emerged as key technologies that have facilitated the discovery of previously unknown details about cell fate specification and gene regulation in the retina. Here we utilized hPSCs integrated with fluorescent reporter genes (SIX6-p2A-eGFP/CRX-p2A-h2b-mRuby3) to generate retinal organoids and carry out bulk RNA sequencing of samples encompassing the majority of retinogenesis (D0-D280). This data set will serve as a valuable reference for the vision research community to characterize differentially expressed genes in the developing human eye.
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Affiliation(s)
- Devansh Agarwal
- Viterbi Family Department of Ophthalmology at the Shiley Eye Institute, University of California San Diego, La Jolla, CA, 92093, USA
- Department of Bioengineering, University of California San Diego, La Jolla, CA, 92093, USA
| | - Rian Kuhns
- Department of Biology, James Madison University, Harrisonburg, VA, 22807, USA
| | | | - Emmalyn Barlow
- Department of Biology, James Madison University, Harrisonburg, VA, 22807, USA
| | - Karl J Wahlin
- Viterbi Family Department of Ophthalmology at the Shiley Eye Institute, University of California San Diego, La Jolla, CA, 92093, USA.
| | - Ray A Enke
- Department of Biology, James Madison University, Harrisonburg, VA, 22807, USA.
- The Center for Genome & Metagenome Studies, James Madison University Harrisonburg, Harrisonburg, VA, 22807, USA.
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Timilsina S, Kirsch-Mangu T, Werth S, Shepard B, Ma T, Villa-Diaz LG. Enhanced self-renewal of human pluripotent stem cells by simulated microgravity. NPJ Microgravity 2022; 8:22. [PMID: 35787634 PMCID: PMC9253108 DOI: 10.1038/s41526-022-00209-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Accepted: 06/22/2022] [Indexed: 11/19/2022] Open
Abstract
A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent stem cells (hPSC) is still lacking. Here, we used a fast-rotating 2-D clinostat to investigate the sµg effect on proliferation, self-renewal, and cell cycle regulation of hPSCs. We observed significant upregulation of protein translation of pluripotent transcription factors in hPSC cultured in sµg compared to cells cultured in 1g conditions. In addition to a significant increase in expression of telomere elongation genes. Differentiation experiments showed that hPSC cultured in sµg condition were less susceptible to differentiation compared to cells in 1g conditions. These results suggest that sµg enhances hPSC self-renewal. Our study revealed that sµg enhanced the cell proliferation of hPSCs by regulating the expression of cell cycle-associated kinases. RNA-seq analysis indicated that in sµg condition the expression of differentiation and development pathways are downregulated, while multiple components of the ubiquitin proteasome system are upregulated, contributing to an enhanced self-renewal of hPSCs. These effects of sµg were not replicated in human fibroblasts. Taken together, our results highlight pathways and mechanisms in hPSCs vulnerable to microgravity that imposes significant impacts on human health and performance, physiology, and cellular and molecular processes.
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Affiliation(s)
- S Timilsina
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - T Kirsch-Mangu
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - S Werth
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - B Shepard
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - T Ma
- Department of Computer Science, Engineering, Oakland University, Rochester, MI, 48309, USA
| | - L G Villa-Diaz
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA. .,Department of Bioengineering, Oakland University, Rochester, MI, 48309, USA.
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Wu H, Tang X, Wang Y, Wang N, Chen Q, Xie J, Liu S, Zhong Z, Qiu Y, Situ P, Zern MA, Wang J, Chen H, Duan Y. Dextran sulfate prevents excess aggregation of human pluripotent stem cells in 3D culture by inhibiting ICAM1 expression coupled with down-regulating E-cadherin through activating the Wnt signaling pathway. Stem Cell Res Ther 2022; 13:218. [PMID: 35619172 PMCID: PMC9137216 DOI: 10.1186/s13287-022-02890-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Accepted: 04/25/2022] [Indexed: 11/29/2022] Open
Abstract
Background Human pluripotent stem cells (hPSCs) have great potential in applications for regenerative medicine and drug development. However, 3D suspension culture systems for clinical-grade hPSC large-scale production have been a major challenge. Accumulating evidence has demonstrated that the addition of dextran sulfate (DS) could prevent excessive adhesion of hPSCs from forming larger aggregates in 3D suspension culture. However, the signaling and molecular mechanisms underlying this phenomenon remain elusive. Methods By using a cell aggregate culture assay and separating big and small aggregates in suspension culture systems, the potential mechanism and downstream target genes of DS were investigated by mRNA sequence analysis, qRT-PCR validation, colony formation assay, and interference assay. Results Since cellular adhesion molecules (CAMs) play important roles in hPSC adhesion and aggregation, we assumed that DS might prevent excess adhesion through affecting the expression of CAMs in hPSCs. As expected, after DS treatment, we found that the expression of CAMs was significantly down-regulated, especially E-cadherin (E-cad) and intercellular adhesion molecule 1 (ICAM1), two highly expressed CAMs in hPSCs. The role of E-cad in the adhesion of hPSCs has been widely investigated, but the function of ICAM1 in hPSCs is hardly understood. In the present study, we demonstrated that ICAM1 exhibited the capacity to promote the adhesion in hPSCs, and this adhesion was suppressed by the treatment with DS. Furthermore, transcriptomic analysis of RNA-seq revealed that DS treatment up-regulated genes related to Wnt signaling resulting in the activation of Wnt signaling in which SLUG, TWIST, and MMP3/7 were highly expressed, and further inhibited the expression of E-cad. Conclusion Our results demonstrated that DS played an important role in controlling the size of hPSC aggregates in 3D suspension culture by inhibiting the expression of ICAM1 coupled with the down-regulation of E-cad through the activation of the Wnt signaling pathway. These results represent a significant step toward developing the expansion of hPSCs under 3D suspension condition in large-scale cultures. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02890-4.
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Affiliation(s)
- Haibin Wu
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China
| | - Xianglian Tang
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China.,Guangxi Key Laboratory of Reproductive Health and Birth Defects Prevention, Guangxi Health Commission Key Laboratory of Precise Diagnosis and Treatment of Genetic Diseases, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, 530003, Guangxi, People's Republic of China.,Genetic and Metabolic Central Laboratory, Guangxi Birth Defects Research and Prevention Institute, Nanning, 530003, Guangxi, People's Republic of China
| | - Yiyu Wang
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China
| | - Ning Wang
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China
| | - Qicong Chen
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China
| | - Jinghe Xie
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China
| | - Shoupei Liu
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China
| | - Zhiyong Zhong
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China
| | - Yaqi Qiu
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China
| | - Ping Situ
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.,School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou, 510180, People's Republic of China
| | - Mark A Zern
- Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA, 95817, USA
| | - Jue Wang
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China.
| | - Honglin Chen
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China. .,National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, 510180, People's Republic of China. .,Guangdong Provincial Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou, 510180, People's Republic of China. .,Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou, 510180, People's Republic of China.
| | - Yuyou Duan
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, No. 382 Waihuan East Road, Suite 406, Higher Education Mega Center, Guangzhou, 510006, People's Republic of China. .,National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, 510180, People's Republic of China. .,Guangdong Provincial Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou, 510180, People's Republic of China. .,Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou, 510180, People's Republic of China.
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10
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Opportunities and challenges in cardiac tissue engineering from an analysis of two decades of advances. Nat Biomed Eng 2022; 6:327-338. [PMID: 35478227 DOI: 10.1038/s41551-022-00885-3] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Accepted: 03/08/2022] [Indexed: 12/20/2022]
Abstract
Engineered human cardiac tissues facilitate progress in regenerative medicine, disease modelling and drug development. In this Perspective, we reflect on the most notable advances in cardiac tissue engineering from the past two decades by analysing pivotal studies and critically examining the most consequential developments. This retrospective analysis led us to identify key milestones and to outline a set of opportunities, along with their associated challenges, for the further advancement of engineered human cardiac tissues.
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11
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Zhang F, Pang C, Zhu H, Chen Y. Timely stimulation of early embryo promotes the acquisition of pluripotency. Cytometry A 2022; 101:682-691. [PMID: 35332996 DOI: 10.1002/cyto.a.24551] [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: 11/15/2021] [Revised: 02/16/2022] [Accepted: 03/15/2022] [Indexed: 11/06/2022]
Abstract
Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are both pluripotent stem cells from early embryos. Another type of pluripotent stem cells, which are similar with EpiSCs and derive from pre-implantation embryos in feeder-free and chemically defined medium containing Activin A and basic fibroblast growth factors (bFGF), is termed as AFSCs. The pluripotency and self-renewal maintenance of ESCs rely on Leukemia inhibitory factor (LIF)/STAT/BMP4/SMAD signaling, while the pluripotency and self-renewal maintenance of EpiSCs and AFSCs rely on bFGF and Activin/Nodal signaling. However, the establishment efficiency of AFSCs lines is low. In this study, we stimulated early embryos by 2i/LIF (CHIR99021 + PD0325901 + LIF) and Activin A + bFGF respectively, to change the cell fate in inner cell mass (ICM). The "fate changed embryos" by 2i/LIF can efficiently produce AFSCs in feeder-free and chemically defined medium, but the efficiency of embryos treated with Activin A + bFGF were poor. The AFSCs from fate-changed embryos share similar molecular characteristics with conventional AFSCs and EpiSCs. Our results suggest that the advanced stimulation of 2i/LIF and the premature stimulation of Activin A + bFGF contribute to capturing the pluripotent stem cells in early embryos, and the FGF/MAPK signaling dominate early embryo development. Our study provides a new approach to capturing pluripotency from pre-implantation embryos.
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Affiliation(s)
- Fengying Zhang
- Southern Medical University Central Laboratory, Southern Medical University, Guangzhou, China
| | - Changmiao Pang
- School of Basic Medical Science, Southern Medical University, Guangzhou, China
| | - Haoyun Zhu
- School of Basic Medical Science, Southern Medical University, Guangzhou, China
| | - Yanglin Chen
- School of Basic Medical Science, Southern Medical University, Guangzhou, China
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12
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Gage E, Agarwal D, Chenault C, Washington-Brown K, Szvetecz S, Jahan N, Wang Z, Jones MK, Zack DJ, Enke RA, Wahlin KJ. Temporal and Isoform-Specific Expression of CTBP2 Is Evolutionarily Conserved Between the Developing Chick and Human Retina. Front Mol Neurosci 2022; 14:773356. [PMID: 35095414 PMCID: PMC8793361 DOI: 10.3389/fnmol.2021.773356] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Accepted: 11/08/2021] [Indexed: 11/13/2022] Open
Abstract
Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.
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Affiliation(s)
- Elizabeth Gage
- Department of Biology, James Madison University, Harrisonburg, VA, United States
| | - Devansh Agarwal
- Department of Bioengineering, University of California, San Diego, La Jolla, CA, United States
- Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California, San Diego, La Jolla, CA, United States
| | - Calvin Chenault
- Department of Biology, James Madison University, Harrisonburg, VA, United States
| | | | - Sarah Szvetecz
- Department of Mathematics & Statistics, James Madison University, Harrisonburg, VA, United States
| | - Nusrat Jahan
- Department of Mathematics & Statistics, James Madison University, Harrisonburg, VA, United States
- The Center for Genome & Metagenome Studies, James Madison University, Harrisonburg, VA, United States
| | - Zixiao Wang
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Melissa K. Jones
- Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California, San Diego, La Jolla, CA, United States
| | - Donald J. Zack
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States
- Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Ray A. Enke
- Department of Biology, James Madison University, Harrisonburg, VA, United States
- The Center for Genome & Metagenome Studies, James Madison University, Harrisonburg, VA, United States
| | - Karl J. Wahlin
- Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California, San Diego, La Jolla, CA, United States
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13
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Amini MS, Naderi MM, Shirazi A, Aminafshar M, Boroujeni SB, Pournourali M, Malekpour A. Bioactive Materials Derived from Menstrual Blood Stem Cells Enhance the Quality of In Vitro Bovine Embryos. Avicenna J Med Biotechnol 2022; 14:287-293. [PMID: 36504564 PMCID: PMC9706245] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 07/11/2022] [Indexed: 12/15/2022] Open
Abstract
Backgrounds The aim of this study was to determine whether the addition of bioactive materials derived from Menstrual Blood Stem Cells (MenSCs) to the oocyte maturation medium may improve the quality of bovine embryos in vitro. Methods MenSCs were collected from 6 healthy women (between 26 and 36 years old) and after 3 days of culture, their bioactive materials were frozen. The bovine Cumulus-Oocyte-Complexes (COCs) were aspirated from ovarian slaughterhouse and the oocytes with more than three layers of cumulus cells were cultured in vitro in media supplemented with (treatment) and without (control) 10% MenSCs' bioactive materials. After IVM/IVF, the presumptive zygotes were cultured for 8 days. Results The blastocyst rate on day 8 in treatment group was higher than control (40.2±1.9 vs. 23±4.2.3, p=0.001). The ratio of Trophectoderm (TE) and Inner Cell Mass (ICM) (ICM/TE) cells was also greater in treatment group compared to control (30.3±2 vs. 14.9±1; p=0.001). The re-expansion of vitrified blastocysts, 24 hours after warming, in treatment group was higher than control (93.3±2.5 vs. 66.2±8.8; p=0.01). The expression of some genes related to pluripotency and implantation (OCT4, CDX2, and IFNT) were increased in treatment group compared to control (p<0/05). Conclusion In conclusion, the addition of MenSCs' bioactive materials during in vitro maturation of bovine oocytes could improve the quantity and quality of bovine IVP embryos. Also, the expression of some genes associated with pluripotency and implantation in the blastocyst would be increased.
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Affiliation(s)
- Mohammad Sobhan Amini
- Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
| | - Mohammad Mehdi Naderi
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran,Corresponding author: Mohammad Mehdi Naderi, DVM, Ph.D., Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, Tel: +98 21 22432020, Fax: +98 21 22432021, E-mail:,
| | - Abolfazl Shirazi
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
| | - Mehdi Aminafshar
- Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Sara Borjian Boroujeni
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
| | - Mostafa Pournourali
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
| | - Ali Malekpour
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
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14
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Zhang GY, Lv ZM, Ma HX, Chen Y, Yuan Y, Sun PX, Feng YQ, Li YW, Lu WJ, Yang YD, Yang C, Yu XL, Wang C, Liang SL, Zhang ML, Li HL, Li WL. Chemical approach to generating long-term self-renewing pMN progenitors from human embryonic stem cells. J Mol Cell Biol 2021; 14:6459209. [PMID: 34893854 PMCID: PMC8872822 DOI: 10.1093/jmcb/mjab076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Revised: 10/24/2021] [Accepted: 12/06/2021] [Indexed: 11/13/2022] Open
Abstract
Spinal cord impairment involving motor neuron degeneration and demyelination can cause life-long disabilities, but effective clinical interventions for restoring neurological functions have yet been developed. In early spinal cord development, neural progenitors in the pMN ('progenitors of motor neurons') domain, defined by the expression of oligodendrocyte transcription factor 2 (OLIG2), in ventral spinal cord first generate motor neurons and then switch the fate to produce myelin-forming oligodendrocytes. Given their differentiation potential, pMN progenitors could be a valuable cell source for cell therapy in relevant neurological conditions such as spinal cord injury. However, fast generation and expansion of pMN progenitors in vitro while conserving their differentiation potential has so far been technically challenging. In this study, based on the chemical screening, we have developed a new recipe for efficient induction of pMN progenitors from human embryonic stem cells. More importantly, these OLIG2+ pMN progenitors can be stably maintained for multiple passages without losing their ability to produce spinal motor neurons and oligodendrocytes rapidly. Our results suggest that these self-renewing pMN progenitors could potentially be useful as a renewable source of cell transplants for spinal cord injury and demyelinating disorders.
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Affiliation(s)
- Guan-Yu Zhang
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Zhu-Man Lv
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Hao-Xin Ma
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Yu Chen
- Changhai Hospital, Second Military Medical University, Shanghai 200433, China
| | - Yuan Yuan
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Ping-Xin Sun
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Yu-Qi Feng
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Ya-Wen Li
- Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.,Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Wen-Jie Lu
- Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.,Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Yu-Dong Yang
- Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.,Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Cheng Yang
- Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK
| | - Xin-Lu Yu
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Chao Wang
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Shu-Long Liang
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Ming-Liang Zhang
- Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.,Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Hui-Liang Li
- Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK
| | - Wen-Lin Li
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China.,Shanghai Key Laboratory of Cell Engineering, Second Military Medical University, Shanghai 200433, China
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15
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Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, Rodríguez-Fernández S, López-Baltar I, Fuentes-Boquete I, Blanco FJ, Díaz-Prado S. Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells. Mol Ther Methods Clin Dev 2021; 23:569-581. [PMID: 34901305 PMCID: PMC8640166 DOI: 10.1016/j.omtm.2021.10.013] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Revised: 09/22/2021] [Accepted: 10/29/2021] [Indexed: 01/23/2023]
Abstract
Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications.
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Affiliation(s)
- Rocío Castro-Viñuelas
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Human Movement Biomechanics Group (HMBG), Tissue Homeostasis and Disease (THD) Lab, Skeletal Biology and Engineering Research Center (SBE), KU Leuven, 3000 Leuven, Belgium.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Clara Sanjurjo-Rodríguez
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain.,Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
| | - María Piñeiro-Ramil
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Silvia Rodríguez-Fernández
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Isidoro López-Baltar
- Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, 15009 A Coruña, Spain
| | - Isaac Fuentes-Boquete
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Francisco J Blanco
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain.,Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain.,Tissular Bioengineering and Cell Therapy Unit (GBTTC-CHUAC), Rheumatology Group, 15006 A Coruña, Galicia, Spain
| | - Silvia Díaz-Prado
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain.,Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain.,Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain.,Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
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16
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Wahlin KJ, Cheng J, Jurlina SL, Jones MK, Dash NR, Ogata A, Kibria N, Ray S, Eldred KC, Kim C, Heng JS, Phillips J, Johnston RJ, Gamm DM, Berlinicke C, Zack DJ. CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids. Front Cell Dev Biol 2021; 9:764725. [PMID: 34869356 PMCID: PMC8635054 DOI: 10.3389/fcell.2021.764725] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Accepted: 10/11/2021] [Indexed: 01/29/2023] Open
Abstract
Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide "retina-like" structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.
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Affiliation(s)
- Karl J. Wahlin
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States,*Correspondence: Karl J. Wahlin,
| | - Jie Cheng
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Shawna L. Jurlina
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Melissa K. Jones
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Nicholas R. Dash
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Anna Ogata
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Nawal Kibria
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Sunayan Ray
- Shiley Eye Institute, University of California, San Diego, San Diego, CA, United States
| | - Kiara C. Eldred
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Catherine Kim
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Jacob S. Heng
- Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States,Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, United States,Department of Ophthalmology and Visual Science, Yale School of Medicine, New Haven, CT, United States
| | - Jenny Phillips
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States
| | - Robert J. Johnston
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - David M. Gamm
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States,Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States,McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, United States
| | - Cynthia Berlinicke
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Donald J. Zack
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States,Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States,Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, United States,Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
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17
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Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties. Cells 2021; 10:cells10112816. [PMID: 34831039 PMCID: PMC8616047 DOI: 10.3390/cells10112816] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 10/13/2021] [Accepted: 10/15/2021] [Indexed: 11/17/2022] Open
Abstract
Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.
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18
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Sordi MB, Cruz A, Fredel MC, Magini R, Sharpe PT. Three-dimensional bioactive hydrogel-based scaffolds for bone regeneration in implant dentistry. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2021; 124:112055. [PMID: 33947549 DOI: 10.1016/j.msec.2021.112055] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 03/11/2021] [Accepted: 03/12/2021] [Indexed: 12/13/2022]
Abstract
Bone tissue requires a range of complex mechanisms to allow the restoration of its structure and function. Bone healing is a signaling cascade process, involving cells secreting cytokines, growth factors, and pro-inflammatory factors in the defect site that will, subsequently, recruit surrounding stem cells to migrate, proliferate, and differentiate into bone-forming cells. Bioactive functional scaffolds could be applied to improve the bone healing processes where the organism is not able to fully regenerate the lost tissue. However, to be optimal, such scaffolds should act as osteoconductors - supporting bone-forming cells, providing nutrients, and sustaining the arrival of new blood vessels, and act as osteoinducers - slowly releasing signaling molecules that stimulate mesenchymal stem cells to differentiate and deposit mineralized bone matrix. Different compositions and shapes of scaffolds, cutting-edge technologies, application of signaling molecules to promote cell differentiation, and high-quality biomaterials are reaching favorable outcomes towards osteoblastic differentiation of stem cells in in vitro and in vivo researches for bone regeneration. Hydrogel-based biomaterials are being pointed as promising for bone tissue regeneration; however, despite all the research and high-impact scientific publications, there are still several challenges that prevent the use of hydrogel-based scaffolds for bone regeneration being feasible for their clinical application. Hence, the objective of this review is to consolidate and report, based on the current scientific literature, the approaches for bone tissue regeneration using bioactive hydrogel-based scaffolds, cell-based therapies, and three-dimensional bioprinting to define the key challenges preventing their use in clinical applications.
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Affiliation(s)
- Mariane B Sordi
- Research Center on Dental Implants, Department of Odontology, Federal University of Santa Catarina, 88040-900 Florianopolis, SC, Brazil; Centre for Craniofacial and Regenerative Biology, Guy's Hospital, King's College London, SE1 9RT, UK.
| | - Ariadne Cruz
- Department of Odontology, Federal University of Santa Catarina, 88040-900 Florianopolis, SC, Brazil.
| | - Márcio C Fredel
- Ceramic and Composite Materials Research Group, Department of Mechanical Engineering, Federal University of Santa Catarina, 88040-900 Florianopolis, SC, Brazil.
| | - Ricardo Magini
- Department of Odontology, Federal University of Santa Catarina, 88040-900 Florianopolis, SC, Brazil
| | - Paul T Sharpe
- Centre for Craniofacial and Regenerative Biology, Guy's Hospital, King's College London, SE1 9RT, UK.
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19
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Generation of high yield insulin-producing cells (IPCs) from various sources of stem cells. VITAMINS AND HORMONES 2021; 116:235-268. [PMID: 33752820 DOI: 10.1016/bs.vh.2021.02.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Type 1 diabetes mellitus occurs when beta cell mass is reduced to less than 20% of the normal level due to immune system destruction of beta cell resulting in an inability to secrete enough insulin. The prevalence of diabetes is expanding according to the American Diabetes Association and the World Health Organization (WHO), foretold to exceed 350 million by 2030. The current treatment does not cure many of the serious complications associated with the disease such as neuropathy, nephropathy, dyslipidemia, retinopathy and cardiovascular disease. Whole pancreas or isolated pancreatic islet transplantation as an alternative therapy can prevent or reduce some of the complications of diabetes. However, the shortage of matched organ or islets cells donor and alloimmune responses limit this therapeutic strategy. Recently, several reports have raised extremely promising results to use different sources of stem cells to differentiate insulin-producing cells and focus on the expansion of these alternative sources. Stem cells, due to their potential for multiple differentiation and self-renewal can differentiate into all cell types, including insulin-producing cells (IPCs). Generation of new beta cells can be achieved from various stem cell sources, including embryonic stem cells (ESCs), adult stem cells, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs). Thus, this chapter discusses on the assistance of cellular reprogramming of various stem cells as candidates for the generation of IPCs using transcription factors/miRNA, cytokines/small molecules and tissue engineering.
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20
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Lu J, Xie ZY, Zhu DH, Li LJ. Human menstrual blood-derived stem cells as immunoregulatory therapy in COVID-19: A case report and review of the literature. World J Clin Cases 2021; 9:1705-1713. [PMID: 33728315 PMCID: PMC7942055 DOI: 10.12998/wjcc.v9.i7.1705] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 12/24/2020] [Accepted: 01/07/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The coronavirus disease 2019 (COVID-19) caused by novel coronavirus 2019 in December 2019 has spread all around the globe and has caused a pandemic. There is still no current effective guidance on the clinical management of COVID-19. Mesenchymal stem cell therapy has been shown to be one of the therapeutic approaches to alleviate pneumonia and symptoms through their immunomo-dulatory effect in COVID-19 patients.
CASE SUMMARY We describe the first confirmed case of COVID-19 in Hangzhou to explore the role of human menstrual blood-derived stem cells (MenSCs) in the treatment of COVID-19. Moreover, we review the immunomodulation effect including non-specific and specific immune functions of MenSCs for the therapy of COVID-19.
CONCLUSION MenSCs can be helpful to find a promising therapeutic approach for COVID-19.
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Affiliation(s)
- Juan Lu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China
| | - Zhong-Yang Xie
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China
| | - Dan-Hua Zhu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China
| | - Lan-Juan Li
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China
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21
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Sarkar A, Saha S, Paul A, Maji A, Roy P, Maity TK. Understanding stem cells and its pivotal role in regenerative medicine. Life Sci 2021; 273:119270. [PMID: 33640402 DOI: 10.1016/j.lfs.2021.119270] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 02/06/2021] [Accepted: 02/14/2021] [Indexed: 02/07/2023]
Abstract
Stem cells (SCs) are clonogenic cells that develop into the specialized cells which later responsible for making up various types of tissue in the human body. SCs are not only the appropriate source of information for cell division, molecular and cellular processes, and tissue homeostasis but also one of the major putative biological aids to diagnose and cure various degenerative diseases. This study emphasises on various research outputs that occurred in the past two decades. This will give brief information on classification, differentiation, detection, and various isolation techniques of SCs. Here, the various signalling pathways which includes WNT, Sonic hedgehog, Notch, BMI1 and C-met pathways and how does it effect on the regeneration of various classes of SCs and factors that regulates the potency of the SCs are also been discussed. We also focused on the application of SCs in the area of regenerative medicine along with the cellular markers that are useful as salient diagnostic or curative tools or in both, by the process of reprogramming, which includes diabetes, cancer, cardiovascular disorders and neurological disorders. The biomarkers that are mentioned in various literatures and experiments include PDX1, FOXA2, HNF6, and NKX6-1 (for diabetes); CD33, CD24, CD133 (for cancer); c-Kit, SCA-1, Wilm's tumor 1 (for cardiovascular disorders); and OCT4, SOX2, c-MYC, EN1, DAT and VMAT2 (for neurological disorders). In this review, we come to know the advancements and scopes of potential SC-based therapies, its diverse applications in clinical fields that can be helpful in the near future.
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Affiliation(s)
- Arnab Sarkar
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Sanjukta Saha
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Abhik Paul
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Avik Maji
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Puspita Roy
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Tapan Kumar Maity
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India.
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22
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Kolagar TA, Farzaneh M, Nikkar N, Khoshnam SE. Human Pluripotent Stem Cells in Neurodegenerative Diseases: Potentials, Advances and Limitations. Curr Stem Cell Res Ther 2020; 15:102-110. [PMID: 31441732 DOI: 10.2174/1574888x14666190823142911] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2019] [Revised: 06/15/2019] [Accepted: 07/17/2019] [Indexed: 12/14/2022]
Abstract
Neurodegenerative diseases are progressive and uncontrolled gradual loss of motor neurons function or death of neuron cells in the central nervous system (CNS) and the mechanisms underlying their progressive nature remain elusive. There is urgent need to investigate therapeutic strategies and novel treatments for neural regeneration in disorders like Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Currently, the development and identification of pluripotent stem cells enabling the acquisition of a large number of neural cells in order to improve cell recovery after neurodegenerative disorders. Pluripotent stem cells which consist of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their ability to indefinitely self-renew and the capacity to differentiate into different types of cells. The first human ESC lines were established from donated human embryos; while, because of a limited supply of donor embryos, human ESCs derivation remains ethically and politically controversial. Hence, hiPSCs-based therapies have been shown as an effective replacement for human ESCs without embryo destruction. Compared to the invasive methods for derivation of human ESCs, human iPSCs has opened possible to reprogram patient-specific cells by defined factors and with minimally invasive procedures. Human pluripotent stem cells are a good source for cell-based research, cell replacement therapies and disease modeling. To date, hundreds of human ESC and human iPSC lines have been generated with the aim of treating various neurodegenerative diseases. In this review, we have highlighted the recent potentials, advances, and limitations of human pluripotent stem cells for the treatment of neurodegenerative disorders.
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Affiliation(s)
- Tannaz Akbari Kolagar
- Faculty of Biological Sciences, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | - Maryam Farzaneh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Negin Nikkar
- Department of Biology, Faculty of Sciences, Alzahra University, Tehran, Iran
| | - Seyed Esmaeil Khoshnam
- Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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23
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Walsh P, Truong V, Nayak S, Saldías Montivero M, Low WC, Parr AM, Dutton JR. Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population. Stem Cells 2020; 38:1400-1408. [PMID: 32745311 PMCID: PMC7693041 DOI: 10.1002/stem.3260] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 06/17/2020] [Accepted: 07/10/2020] [Indexed: 12/22/2022]
Abstract
Differentiation of human pluripotent stem cells (hPSCs) into ectoderm provides neurons and glia useful for research, disease modeling, drug discovery, and potential cell therapies. In current protocols, hPSCs are traditionally differentiated into an obligate rostro-dorsal ectodermal fate expressing PAX6 after 6 to 12 days in vitro when protected from mesendoderm inducers. This rate-limiting step has performed a long-standing role in hindering the development of rapid differentiation protocols for ectoderm-derived cell types, as any protocol requires 6 to 10 days in vitro to simply initiate. Here, we report efficient differentiation of hPSCs into a naive early ectodermal intermediate within 24 hours using combined inhibition of bone morphogenic protein and fibroblast growth factor signaling. The induced population responds immediately to morphogen gradients to upregulate rostro-caudal neurodevelopmental landmark gene expression in a generally accelerated fashion. This method can serve as a new platform for the development of novel, rapid, and efficient protocols for the manufacture of hPSC-derived neural lineages.
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Affiliation(s)
- Patrick Walsh
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
- Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Vincent Truong
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
- Department of Ophthalmology and Visual NeurosciencesUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Sushmita Nayak
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
| | | | - Walter C. Low
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
- Department of NeurosurgeryUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Ann M. Parr
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
- Department of NeurosurgeryUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - James R. Dutton
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMinnesotaUSA
- Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisMinnesotaUSA
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24
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Novel WT1 Target Genes: IL-2, IL-2RB, and IL-2RG Discovered during WT1 Silencing Using Lentiviral-Based RNAi in Myeloid Leukemia Cells. BIOMED RESEARCH INTERNATIONAL 2020; 2020:7851414. [PMID: 33110919 PMCID: PMC7582064 DOI: 10.1155/2020/7851414] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Revised: 09/18/2020] [Accepted: 09/28/2020] [Indexed: 12/19/2022]
Abstract
Wilms' tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms' tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0%, 44 ± 9.5%, and 88 ± 9.1% at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.
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25
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Oss-Ronen L, Redden RA, Lelkes PI. Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity. Stem Cells Dev 2020; 29:1275-1284. [PMID: 32731794 DOI: 10.1089/scd.2020.0097] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.
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Affiliation(s)
- Liat Oss-Ronen
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
| | - Robert A Redden
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
| | - Peter I Lelkes
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
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26
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Topal T, Fan Z, Deng LY, Krebsbach PH, Deng CX. Integrin-Targeted Cyclic Forces Accelerate Neural Tube-Like Rosette Formation from Human Embryonic Stem Cells. ACTA ACUST UNITED AC 2020; 3:e1900064. [PMID: 32648720 DOI: 10.1002/adbi.201900064] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 07/29/2019] [Indexed: 11/11/2022]
Abstract
Mechanical forces play important roles in human embryonic stem cell (hESC) differentiation. To investigate the impact of dynamic mechanical forces on neural induction of hESCs, this study employs acoustic tweezing cytometry (ATC) to apply cyclic forces/strains to hESCs by actuating integrin-bound microbubbles using ultrasound pulses. Accelerated neural induction of hESCs is demonstrated as the result of combined action of ATC and neural induction medium (NIM). Specifically, application of ATC for 30 min followed by culture in NIM upregulates neuroecdoderm markers Pax6 and Sox1 as early as 6 h after ATC, and induces neural tube-like rosette formation at 48 h after ATC. In contrast, no changes are observed in hESCs cultured in NIM without ATC treatment. In the absence of NIM, ATC application decreases Oct4, but does not increase Pax6 and Sox1 expression, nor does it induce neural rossette formation. The effects of ATC are abolished by inhibition of FAK, myosin activity, and RhoA/ROCK signaling. Taken together, the results reveal a synergistic action of ATC and NIM as an integrated mechanobiology mechanism that requires both integrin-targeted cyclic forces and chemical factors for accelerated neural induction of hESCs.
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Affiliation(s)
- Tuğba Topal
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48105, USA.,Biointerfaces Institute, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Zhenzhen Fan
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48105, USA
| | - Laura Y Deng
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Paul H Krebsbach
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48105, USA.,Biointerfaces Institute, University of Michigan, Ann Arbor, MI, 48109, USA.,UCLA School of Dentistry, Los Angeles, CA, 90095, USA
| | - Cheri X Deng
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48105, USA.,Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
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27
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Khalil AS, Xie AW, Johnson HJ, Murphy WL. Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion. Biomaterials 2020; 248:120007. [PMID: 32302801 PMCID: PMC8445021 DOI: 10.1016/j.biomaterials.2020.120007] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2019] [Revised: 02/24/2020] [Accepted: 03/24/2020] [Indexed: 12/17/2022]
Abstract
Translation of human pluripotent stem cell (hPSC)-derived therapies to the clinic demands scalable, cost-effective methods for cell expansion. Culture media currently used for hPSC expansion rely on high concentrations and frequent supplementation of recombinant growth factors due to their short half-life at physiological temperatures. Here, we developed a biomaterial strategy using mineral-coated microparticles (MCMs) to sustain delivery of basic fibroblast growth factor (bFGF), a thermolabile protein critical for hPSC pluripotency and proliferation. We show that the MCMs stabilize bFGF against thermally induced activity loss and provide more efficient sustained release of active growth factor compared to polymeric carriers commonly used for growth factor delivery. Using a statistically driven optimization approach called Design of Experiments, we generated a bFGF-loaded MCM formulation that supported hPSC expansion over 25 passages without the need for additional bFGF supplementation to the media, resulting in greater than 80% reduction in bFGF usage compared to standard approaches. This materials-based strategy to stabilize and sustain delivery of a thermolabile growth factor has broad potential to reduce costs associated with recombinant protein supplements in scalable biomanufacturing of emerging cell therapies.
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Affiliation(s)
- Andrew S Khalil
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Angela W Xie
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Hunter J Johnson
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - William L Murphy
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53705, USA; Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI, 53705, USA; Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI, 53705, USA.
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28
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van Gorp PRR, Trines SA, Pijnappels DA, de Vries AAF. Multicellular In vitro Models of Cardiac Arrhythmias: Focus on Atrial Fibrillation. Front Cardiovasc Med 2020; 7:43. [PMID: 32296716 PMCID: PMC7138102 DOI: 10.3389/fcvm.2020.00043] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Accepted: 03/06/2020] [Indexed: 12/13/2022] Open
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.
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Affiliation(s)
| | | | | | - Antoine A. F. de Vries
- Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Leiden, Netherlands
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29
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Ullah I, Lee R, Oh KB, Hwang S, Kim Y, Hur TY, Ock SA. Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic β-like cells by microenvironment modulation. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2020; 33:1837-1847. [PMID: 32106662 PMCID: PMC7649066 DOI: 10.5713/ajas.19.0796] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Accepted: 01/15/2020] [Indexed: 11/27/2022]
Abstract
OBJECTIVE To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. METHODS The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. RESULTS The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. CONCLUSION 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.
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Affiliation(s)
- Imran Ullah
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea.,Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
| | - Ran Lee
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
| | - Keon Bong Oh
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
| | - Seongsoo Hwang
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
| | - Youngim Kim
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
| | - Tai-Young Hur
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
| | - Sun A Ock
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea
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30
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Bardakova K, Grebenik E, Minaev N, Churbanov S, Moldagazyeva Z, Krupinov G, Kostjuk S, Timashev P. Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 107:110300. [DOI: 10.1016/j.msec.2019.110300] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 09/12/2019] [Accepted: 10/09/2019] [Indexed: 11/26/2022]
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31
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Liu W, Deng C, Godoy-Parejo C, Zhang Y, Chen G. Developments in cell culture systems for human pluripotent stem cells. World J Stem Cells 2019; 11:968-981. [PMID: 31768223 PMCID: PMC6851012 DOI: 10.4252/wjsc.v11.i11.968] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 08/21/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications.
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Affiliation(s)
- Weiwei Liu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Bioimaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Chunhao Deng
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Carlos Godoy-Parejo
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Yumeng Zhang
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China
| | - Guokai Chen
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau 999078, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau 999078, China.
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Kim YH, Kim BJ, Kim SM, Kim SU, Ryu BY. Induction of cardiomyocyte‑like cells from hair follicle cells in mice. Int J Mol Med 2019; 43:2230-2240. [PMID: 30864673 DOI: 10.3892/ijmm.2019.4133] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 03/08/2019] [Indexed: 11/05/2022] Open
Abstract
Hair follicles (HFs) are a well‑characterized niche for adult stem cells (SCs), and include epithelial and melanocytic SCs. HF cells are an accessible source of multipotent adult SCs for the generation of the interfollicular epidermis, HF structures and sebaceous glands in addition to the reconstitution of novel HFs in vivo. In the present study, it was demonstrated that HF cells are able to be induced to differentiate into cardiomyocyte‑like cells in vitro under specific conditions. It was determined that HF cells cultured on OP9 feeder cells in KnockOut‑Dulbecco's modified Eagle's medium/B27 in the presence of vascular endothelial growth factors differentiated into cardiomyocyte‑like cells that express markers specific to cardiac lineage, but do not express non‑cardiac lineage markers including neural stem/progenitor cell, HF bulge cells or undifferentiated spermatogonia markers. These cardiomyocyte‑like cells exhibited a spindle‑ and filament‑shaped morphology similar to that presented by cardiac muscles and exhibited spontaneous beating that persisted for over 3 months. These results demonstrate that SC reprogramming and differentiation may be induced without resulting in any genetic modification, which is important for the clinical applications of SCs including tissue and organ regeneration.
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Affiliation(s)
- Yong-Hee Kim
- Department of Animal Science and Technology, College of Biotechnology and Natural Resources, Chung‑Ang University, Anseong, Gyeonggi‑do 17546, Republic of Korea
| | - Bang-Jin Kim
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Seok-Man Kim
- Department of Animal Science and Technology, College of Biotechnology and Natural Resources, Chung‑Ang University, Anseong, Gyeonggi‑do 17546, Republic of Korea
| | - Sun-Uk Kim
- National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungcheongbuk‑do 28116, Republic of Korea
| | - Buom-Yong Ryu
- Department of Animal Science and Technology, College of Biotechnology and Natural Resources, Chung‑Ang University, Anseong, Gyeonggi‑do 17546, Republic of Korea
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Ota M, Takagaki K, Takaoka S, Tanemura H, Urushihata N. A new method to confirm the absence of human and animal serum in mesenchymal stem cell culture media. Int J Med Sci 2019; 16:1102-1106. [PMID: 31523172 PMCID: PMC6743277 DOI: 10.7150/ijms.32100] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/08/2018] [Accepted: 03/01/2019] [Indexed: 01/19/2023] Open
Abstract
Mesenchymal stem cells are an ideal source for regenerative medicine. For clinical use, cell culture should be done at stable conditions, thus the use of serum should be avoided because of the batch-to-batch variations of serum. Although several kinds of serum-free media are available, a method to confirm whether they contain serum has not been established yet. During studies on effect of adipocyte mesenchymal stem cells (Ad-MSCs) on pain using a human pain gene array, we noticed that BDKRB1 gene was constantly upregulated when serum was used in the culture medium. In this study, we attempted to establish further the potential of this gene as a new marker indicative of the presence of serum in media. Using a real-time quantitative PCR gene array screening containing 84 functional genes, we verified BDKRB1 as a specific gene upregulated in the presence of serum. The expression of BDKRB1 in Ad-MSCs was induced not only by bovine serum but also by human serum. The BDKRB1 expression was induced even when Ad-MSCs was cultured with 0.1% serum in the medium. We concluded that BDKRB1 is a valuable marker to detect traces of both human and animal serum in Ad-MSCs cultures. Our study provides a new method to confirm the absence of serum in media and ensure a stable cell culture condition.
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Affiliation(s)
- Megumi Ota
- BioMimetics Sympathies Inc., Aomi, Koto-Ku, Tokyo, Japan
| | | | - Sho Takaoka
- BioMimetics Sympathies Inc., Aomi, Koto-Ku, Tokyo, Japan
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34
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Haghighi F, Dahlmann J, Nakhaei-Rad S, Lang A, Kutschka I, Zenker M, Kensah G, Piekorz RP, Ahmadian MR. bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling. Cell Commun Signal 2018; 16:96. [PMID: 30518391 PMCID: PMC6282345 DOI: 10.1186/s12964-018-0307-1] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2018] [Accepted: 11/21/2018] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs. METHODS bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation. RESULTS Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs. CONCLUSION Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.
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Affiliation(s)
- Fereshteh Haghighi
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany
| | - Julia Dahlmann
- Department of Thoracic and Cardiovascular Surgery, University of Göttingen, Göttingen, Germany.,Department of Cardiothoracic Surgery, University Clinic, Otto von Guericke-University, Magdeburg, Germany
| | - Saeideh Nakhaei-Rad
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany
| | - Alexander Lang
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany.,Present address: Department of Urology, Medical Faculty of Heinrich Heine University, Düsseldorf, Germany
| | - Ingo Kutschka
- Department of Cardiothoracic Surgery, University Clinic, Otto von Guericke-University, Magdeburg, Germany
| | - Martin Zenker
- Institute of Human Genetics, Otto von Guericke-University, Magdeburg, Germany
| | - George Kensah
- Department of Thoracic and Cardiovascular Surgery, University of Göttingen, Göttingen, Germany.,Department of Cardiothoracic Surgery, University Clinic, Otto von Guericke-University, Magdeburg, Germany
| | - Roland P Piekorz
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany
| | - Mohammad Reza Ahmadian
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany.
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Dakhore S, Nayer B, Hasegawa K. Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement. Stem Cells Int 2018; 2018:7396905. [PMID: 30595701 PMCID: PMC6282144 DOI: 10.1155/2018/7396905] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2018] [Revised: 10/23/2018] [Accepted: 10/24/2018] [Indexed: 12/23/2022] Open
Abstract
Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.
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Affiliation(s)
- Sushrut Dakhore
- Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences (NCBS), Bangalore, India
| | - Bhavana Nayer
- Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences (NCBS), Bangalore, India
| | - Kouichi Hasegawa
- Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences (NCBS), Bangalore, India
- Institute for Integrated Cell-Material Sciences (iCeMS), Institute for Advanced Study, Kyoto University, Japan
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36
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Isolation, Culture, and Functional Characterization of Human Embryonic Stem Cells: Current Trends and Challenges. Stem Cells Int 2018; 2018:1429351. [PMID: 30254679 PMCID: PMC6142731 DOI: 10.1155/2018/1429351] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Revised: 07/19/2018] [Accepted: 07/30/2018] [Indexed: 12/12/2022] Open
Abstract
Human embryonic stem cells (hESCs) hold great potential for the treatment of various degenerative diseases. Pluripotent hESCs have a great ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in the body. The journey of hESC research is not that smooth, as it has faced several challenges which are limited to not only tumor formation and immunorejection but also social, ethical, and political aspects. The isolation of hESCs from the human embryo is considered highly objectionable as it requires the destruction of the human embryo. The issue was debated and discussed in both public and government platforms, which led to banning of hESC research in many countries around the world. The banning has negatively affected the progress of hESC research as many federal governments around the world stopped research funding. Afterward, some countries lifted the ban and allowed the funding in hESC research, but the damage has already been done on the progress of research. Under these unfavorable conditions, still some progress was made to isolate, culture, and characterize hESCs using different strategies. In this review, we have summarized various strategies used to successfully isolate, culture, and characterize hESCs. Finally, hESCs hold a great promise for clinical applications with proper strategies to minimize the teratoma formation and immunorejection and better cell transplantation strategies.
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Comparative Assessment on the Expression Level of Recombinant Human Follicle-Stimulating Hormone (FSH) in Serum-Containing Versus Protein-Free Culture Media. Mol Biotechnol 2018; 59:490-498. [PMID: 28993982 DOI: 10.1007/s12033-017-0037-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media. The expression cassette entailing FSH subunits was transfected to CHO/dhfr- and CHO DG44 cell lines, and gene amplification was achieved using dihydrofolate reductase (DHFR)/methotrexate (MTX) system. Afterward, the expression level of rhFSH was studied using real-time PCR, Western blotting and ELISA. Our achievements revealed that stepwise increase in MTX [up to 2000 nano-molar (nM)] leads to boost the expression level of rhFSH mRNA in both cell lines, although DG44 have better results, as mRNA expression level reached 124.8- and 168.3-fold in alpha and beta subunits, respectively. DG44 cells have also the best protein production in 2000 nM MTX, which reached 1.7-fold in comparison with that of the mock group. According to the above results and many advantages of protein-free media, DG44 is preferable cell line for future steps.
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38
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Rattananinsruang P, Dechsukhum C, Leeanansaksiri W. Establishment of Insulin-Producing Cells From Human Embryonic Stem Cells Underhypoxic Condition for Cell Based Therapy. Front Cell Dev Biol 2018; 6:49. [PMID: 29868580 PMCID: PMC5962719 DOI: 10.3389/fcell.2018.00049] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 04/16/2018] [Indexed: 12/27/2022] Open
Abstract
Diabetes mellitus (DM) is a group of diseases characterized by abnormally high levels of glucose in the blood stream. In developing a potential therapy for diabetic patients, pancreatic cells transplantation has drawn great attention. However, the hinder of cell transplantation for diabetes treatment is insufficient sources of insulin-producing cells. Therefore, new cell based therapy need to be developed. In this regard, human embryonic stem cells (hESCs) may serve as good candidates for this based on their capability of differentiation into various cell types. In this study, we designed a new differentiation protocol that can generate hESC-derived insulin-producing cells (hES-DIPCs) in a hypoxic condition. We also emphasized on the induction of definitive endoderm during embryoid bodies (EBs) formation. After induction of hESCs differentiation into insulin-producing cells (IPCs), the cells obtained from the cultures exhibited pancreas-related genes such as Pdx1, Ngn3, Nkx6.1, GLUT2, and insulin. These cells also showed positive for DTZ-stained cellular clusters and contained ability of insulin secretion in a glucose-dependent manner. After achievement to generated functional hES-DIPCs in vitro, some of the hES-DIPCs were then encapsulated named encapsulated hES-DIPCs. The data showed that the encapsulated cells could possess the function of insulin secretion in a time-dependent manner. The hES-DIPCs and encapsulated hES-DIPCs were then separately transplanted into STZ-induced diabetic mice. The findings showed the significant blood glucose levels regulation capacity and declination of IL-1β concentration in all transplanted mice. These results indicated that both hES-DIPCs and encapsulated hES-DIPCs contained the ability to sustain hyperglycemia condition as well as decrease inflammatory cytokine level in vivo. The findings of this study may apply for generation of a large number of hES-DIPCs in vitro. In addition, the implication of this work is therapeutic value in type I diabetes treatment in the future. The application for type II diabetes treatment remain to be investigated.
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Affiliation(s)
- Piyaporn Rattananinsruang
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Chavaboon Dechsukhum
- School of Pathology, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Wilairat Leeanansaksiri
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
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Magdy T, Schuldt AJT, Wu JC, Bernstein D, Burridge PW. Human Induced Pluripotent Stem Cell (hiPSC)-Derived Cells to Assess Drug Cardiotoxicity: Opportunities and Problems. Annu Rev Pharmacol Toxicol 2018; 58:83-103. [PMID: 28992430 PMCID: PMC7386286 DOI: 10.1146/annurev-pharmtox-010617-053110] [Citation(s) in RCA: 78] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Billions of US dollars are invested every year by the pharmaceutical industry in drug development, with the aim of introducing new drugs that are effective and have minimal side effects. Thirty percent of in-pipeline drugs are excluded in an early phase of preclinical and clinical screening owing to cardiovascular safety concerns, and several lead molecules that pass the early safety screening make it to market but are later withdrawn owing to severe cardiac side effects. Although the current drug safety screening methodologies can identify some cardiotoxic drug candidates, they cannot accurately represent the human heart in many aspects, including genomics, transcriptomics, and patient- or population-specific cardiotoxicity. Despite some limitations, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful and evolving technology that has been shown to recapitulate many attributes of human cardiomyocytes and their drug responses. In this review, we discuss the potential impact of the inclusion of the hiPSC-CM platform in premarket candidate drug screening.
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Affiliation(s)
- Tarek Magdy
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA;
- Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
| | - Adam J T Schuldt
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA;
- Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
- Division of Cardiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
| | - Joseph C Wu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305, USA
- Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Daniel Bernstein
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305, USA
- Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Paul W Burridge
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA;
- Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
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Liu X, Su P, Lu L, Feng Z, Wang H, Zhou J. Function of FEZF1 during early neural differentiation of human embryonic stem cells. SCIENCE CHINA-LIFE SCIENCES 2018; 61:35-45. [PMID: 29318501 DOI: 10.1007/s11427-017-9155-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/13/2017] [Accepted: 11/19/2017] [Indexed: 12/14/2022]
Abstract
The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study, using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover, loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs, suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.
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Affiliation(s)
- Xin Liu
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China
| | - Pei Su
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China
| | - Lisha Lu
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China
| | - Zicen Feng
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China
| | - Hongtao Wang
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China.
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China.
| | - Jiaxi Zhou
- State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China.
- Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, 300000, China.
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Chandrasekaran A, Avci HX, Ochalek A, Rösingh LN, Molnár K, László L, Bellák T, Téglási A, Pesti K, Mike A, Phanthong P, Bíró O, Hall V, Kitiyanant N, Krause KH, Kobolák J, Dinnyés A. Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells. Stem Cell Res 2017; 25:139-151. [PMID: 29128818 DOI: 10.1016/j.scr.2017.10.010] [Citation(s) in RCA: 90] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Revised: 10/06/2017] [Accepted: 10/10/2017] [Indexed: 02/06/2023] Open
Abstract
Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.
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Affiliation(s)
- Abinaya Chandrasekaran
- BioTalentum Ltd, Gödöllő, Hungary; Molecular Animal Biotechnology Lab, Szent István University, Gödöllő, Hungary
| | - Hasan X Avci
- BioTalentum Ltd, Gödöllő, Hungary; Department of Anatomy, Embryology and Histology, Faculty of Medicine, University of Szeged, Szeged, Hungary
| | - Anna Ochalek
- BioTalentum Ltd, Gödöllő, Hungary; Molecular Animal Biotechnology Lab, Szent István University, Gödöllő, Hungary
| | - Lone N Rösingh
- Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland
| | - Kinga Molnár
- Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary
| | - Lajos László
- Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary
| | - Tamás Bellák
- BioTalentum Ltd, Gödöllő, Hungary; Department of Anatomy, Embryology and Histology, Faculty of Medicine, University of Szeged, Szeged, Hungary
| | | | - Krisztina Pesti
- Opto-Neuropharmacology Group, MTA-ELTE NAP B, Budapest, Hungary; János Szentágothai Doctoral School of Neurosciences, Semmelweis University, Budapest, Hungary
| | - Arpad Mike
- Opto-Neuropharmacology Group, MTA-ELTE NAP B, Budapest, Hungary
| | - Phetcharat Phanthong
- BioTalentum Ltd, Gödöllő, Hungary; Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom Bangkok, Thailand
| | - Orsolya Bíró
- First Department of Obstetrics and Gynaecology, Semmelweis University, Budapest, Hungary
| | - Vanessa Hall
- Department of Veterinary and Animal Science, University of Copenhagen, Denmark
| | - Narisorn Kitiyanant
- Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom Bangkok, Thailand
| | - Karl-Heinz Krause
- Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland
| | | | - András Dinnyés
- BioTalentum Ltd, Gödöllő, Hungary; Molecular Animal Biotechnology Lab, Szent István University, Gödöllő, Hungary.
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Son B, Kim JA, Cho S, Jeong GJ, Kim BS, Hwang NS, Park TH. Lineage Specific Differentiation of Magnetic Nanoparticle-Based Size Controlled Human Embryoid Body. ACS Biomater Sci Eng 2017; 3:1719-1729. [DOI: 10.1021/acsbiomaterials.7b00141] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Affiliation(s)
- Boram Son
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
| | - Jeong Ah Kim
- Biomedical
Omics Group, Korea Basic Science Institute, Cheongju, Chungbuk 28119, Republic of Korea
| | - Sungwoo Cho
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
| | - Gun-Jae Jeong
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
| | - Byung Soo Kim
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
| | - Nathaniel S. Hwang
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
| | - Tai Hyun Park
- School
of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
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Omami M, McGarrigle JJ, Reedy M, Isa D, Ghani S, Marchese E, Bochenek MA, Longi M, Xing Y, Joshi I, Wang Y, Oberholzer J. Islet Microencapsulation: Strategies and Clinical Status in Diabetes. Curr Diab Rep 2017; 17:47. [PMID: 28523592 DOI: 10.1007/s11892-017-0877-0] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
PURPOSE OF REVIEW Type 1 diabetes mellitus (T1DM) is an autoimmune disease that results from the destruction of insulin-producing pancreatic β cells in the islets of Langerhans. Islet cell transplantation has become a successful therapy for specific patients with T1DM with hypoglycemic unawareness. The reversal of T1DM by islet transplantation is now performed at many major medical facilities throughout the world. However, many challenges must still be overcome in order to achieve continuous, long-term successful transplant outcomes. Two major obstacles to this therapy are a lack of islet cells for transplantation and the need for life-long immunosuppressive treatment. Microencapsulation is seen as a technology that can overcome both these limitations of islet cell transplantation. This review depicts the present state of microencapsulated islet transplantation. RECENT FINDINGS Microencapsulation can play a significant role in overcoming the need for immunosuppression and lack of donor islet cells. This review focuses on microencapsulation and the clinical status of the technology in combating T1DM.
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Affiliation(s)
- Mustafa Omami
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - James J McGarrigle
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA.
| | - Mick Reedy
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Douglas Isa
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Sofia Ghani
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Enza Marchese
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Matthew A Bochenek
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
- Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA
| | - Maha Longi
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Yuan Xing
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
- Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA
| | - Ira Joshi
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
| | - Yong Wang
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
- Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA
| | - José Oberholzer
- Department of Surgery, University of Illinois at Chicago, 840 S. Wood St. Room 502, Chicago, IL, 60612, USA
- Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA
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44
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Jeong HC, Park SJ, Choi JJ, Go YH, Hong SK, Kwon OS, Shin JG, Kim RK, Lee MO, Lee SJ, Shin HD, Moon SH, Cha HJ. PRMT8 Controls the Pluripotency and Mesodermal Fate of Human Embryonic Stem Cells By Enhancing the PI3K/AKT/SOX2 Axis. Stem Cells 2017; 35:2037-2049. [DOI: 10.1002/stem.2642] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 05/06/2017] [Indexed: 12/31/2022]
Affiliation(s)
- Ho-Chang Jeong
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Soon-Jung Park
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Jong-Jin Choi
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Young-Hyun Go
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Soon-Ki Hong
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Ok-Seon Kwon
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
| | - Joong-Gon Shin
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
- Research Institute for Basic Science, Sogang University; Seoul Republic of Korea
| | - Rae-Kwon Kim
- Department of Life Science; Research Institute for Natural Sciences, Hanyang University; Seoul Republic of Korea
| | - Mi-Ok Lee
- Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon Republic of Korea
| | - Su-Jae Lee
- Department of Life Science; Research Institute for Natural Sciences, Hanyang University; Seoul Republic of Korea
| | - Hyoung Doo Shin
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
- Research Institute for Basic Science, Sogang University; Seoul Republic of Korea
| | - Sung-Hwan Moon
- Department of Medicine; School of Medicine, Konkuk University; Seoul Republic of Korea
| | - Hyuk-Jin Cha
- Department of Life Sciences, Sogang University; Seoul Republic of Korea
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45
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Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells. Sci Rep 2017; 7:766. [PMID: 28396597 PMCID: PMC5429674 DOI: 10.1038/s41598-017-00774-9] [Citation(s) in RCA: 182] [Impact Index Per Article: 22.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 03/14/2017] [Indexed: 12/22/2022] Open
Abstract
The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.
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Abstract
Background Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal‐cell culture media. Methods A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. Results At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical‐based synthetic media because naturally derived ingredients have their disadvantages such as large batch‐to‐batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum‐containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Conclusions Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.
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Affiliation(s)
- Tatsuma Yao
- Research and Development Center Fuso Pharmaceutical Industries, Ltd. Osaka Japan.,Faculty of Biology-Oriented Science and Technology Kindai University Wakayama Japan
| | - Yuta Asayama
- Research and Development Center Fuso Pharmaceutical Industries, Ltd. Osaka Japan
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47
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Nishimura T, Hatoya S, Kanegi R, Wijesekera DPH, Sanno K, Tanaka E, Sugiura K, Hiromitsu Tamada NK, Imai H, Inaba T. Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions. Mol Reprod Dev 2017; 84:329-339. [PMID: 28240438 DOI: 10.1002/mrd.22789] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 02/09/2017] [Indexed: 12/29/2022]
Abstract
Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.
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Affiliation(s)
- Toshiya Nishimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Ryoji Kanegi
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | | | - Kousuke Sanno
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Erina Tanaka
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | | | - Hiroshi Imai
- Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Toshio Inaba
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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48
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Matsuoka AJ, Morrissey ZD, Zhang C, Homma K, Belmadani A, Miller CA, Chadly DM, Kobayashi S, Edelbrock AN, Tanaka‐Matakatsu M, Whitlon DS, Lyass L, McGuire TL, Stupp SI, Kessler JA. Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons. Stem Cells Transl Med 2017; 6:923-936. [PMID: 28186679 PMCID: PMC5442760 DOI: 10.1002/sctm.16-0032] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2016] [Revised: 08/31/2016] [Accepted: 10/19/2016] [Indexed: 12/15/2022] Open
Abstract
The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.
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Affiliation(s)
- Akihiro J. Matsuoka
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
- Department of Communication Sciences and DisordersChicagoILUSA
- Knowles Hearing CenterChicagoILUSA
| | | | - Chaoying Zhang
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
| | - Kazuaki Homma
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
- Knowles Hearing CenterChicagoILUSA
| | - Abdelhak Belmadani
- Department of Molecular Pharmacology and Biological ChemistryChicagoILUSA
| | | | - Duncan M. Chadly
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
| | - Shun Kobayashi
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
| | | | | | - Donna S. Whitlon
- Department of Otolaryngology and Head and Neck SurgeryChicagoILUSA
- Knowles Hearing CenterChicagoILUSA
| | - Ljuba Lyass
- Department of Biomedical EngineeringChicagoILUSA
| | | | - Samuel I. Stupp
- Department of MedicineChicagoILUSA
- Department of Biomedical EngineeringChicagoILUSA
- Simpson Querrey Institute for BioNanotechnologyChicagoILUSA
- Department of ChemistryNorthwestern University
- Department of Materials Science & EngineeringNorthwestern University
| | - John A. Kessler
- Department of NeurologyFeinberg School of Medicine, Northwestern UniversityChicagoILUSA
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49
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Fan Z, Liu L, Huang X, Zhao Y, Zhou L, Wang D, Wei J. Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition. Dev Growth Differ 2017; 59:83-93. [PMID: 28230233 DOI: 10.1111/dgd.12341] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Revised: 01/10/2017] [Accepted: 01/17/2017] [Indexed: 01/02/2023]
Abstract
Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.
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Affiliation(s)
- Zhenhua Fan
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Linyan Liu
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Xiaohuan Huang
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Yang Zhao
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Linyan Zhou
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Deshou Wang
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
| | - Jing Wei
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China
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50
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McDaniel K, Meng F, Wu N, Sato K, Venter J, Bernuzzi F, Invernizzi P, Zhou T, Kyritsi K, Wan Y, Huang Q, Onori P, Francis H, Gaudio E, Glaser S, Alpini G. Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice‡. Hepatology 2017; 65:544-559. [PMID: 27639079 PMCID: PMC5258713 DOI: 10.1002/hep.28831] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/07/2016] [Revised: 08/01/2016] [Accepted: 09/01/2016] [Indexed: 12/14/2022]
Abstract
UNLABELLED Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/- ) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. CONCLUSION The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary-committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544-559).
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Affiliation(s)
- Kelly McDaniel
- Research, Central Texas Veterans Health Care System, Temple, TX, USA,Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA,Research Institute, Baylor Scott & White Health, Temple, TX, USA
| | - Fanyin Meng
- Research, Central Texas Veterans Health Care System, Temple, TX, USA,Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA,Research Institute, Baylor Scott & White Health, Temple, TX, USA
| | - Nan Wu
- Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Keisaku Sato
- Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Julie Venter
- Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Francesca Bernuzzi
- Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Milan, Italy
| | - Pietro Invernizzi
- Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Milan, Italy
| | - Tianhao Zhou
- Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Konstantina Kyritsi
- Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Ying Wan
- Research Institute, Baylor Scott & White Health, Temple, TX, USA,Department of Pathophysiology, Key Lab for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou, China
| | - Qiaobing Huang
- Department of Pathophysiology, Key Lab for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou, China
| | - Paolo Onori
- Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy
| | - Heather Francis
- Research, Central Texas Veterans Health Care System, Temple, TX, USA,Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA,Research Institute, Baylor Scott & White Health, Temple, TX, USA
| | - Eugenio Gaudio
- Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy
| | - Shannon Glaser
- Research, Central Texas Veterans Health Care System, Temple, TX, USA,Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
| | - Gianfranco Alpini
- Research, Central Texas Veterans Health Care System, Temple, TX, USA,Department of Medicine, Baylor Scott & White Health Digestive Disease Research Center, Texas A&M HSC and Baylor Scott & White Health, Temple, TX, USA
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