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Nityanandam A, Patton MH, Bayazitov IT, Newman KD, Thomas KT, Zakharenko SS. Protocol for generating human assembloids to investigate thalamocortical and corticothalamic synaptic transmission and plasticity. STAR Protoc 2025; 6:103630. [PMID: 39921865 PMCID: PMC11850219 DOI: 10.1016/j.xpro.2025.103630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2024] [Revised: 12/05/2024] [Accepted: 01/15/2025] [Indexed: 02/10/2025] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) can be used to generate assembloids that recreate thalamocortical circuitry displaying short-term and long-term synaptic plasticity. Here, we describe a protocol for differentiating hiPSCs into thalamic and cortical organoids and then fusing them to generate thalamocortical assembloids. We detail the steps for using whole-cell patch-clamp electrophysiology to investigate the properties of synaptic transmission and synaptic plasticity in this model system. For complete details on the use and execution of this protocol, please refer to Patton et al.1.
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Affiliation(s)
- Anjana Nityanandam
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
| | - Mary H Patton
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Ildar T Bayazitov
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Kyle D Newman
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Kristen T Thomas
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Stanislav S Zakharenko
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
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Yang HS, Zheng YX, Bai X, He XY, Wang TH. Application prospects of urine-derived stem cells in neurological and musculoskeletal diseases. World J Orthop 2024; 15:918-931. [PMID: 39473520 DOI: 10.5312/wjo.v15.i10.918] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2024] [Revised: 08/25/2024] [Accepted: 09/09/2024] [Indexed: 10/11/2024] Open
Abstract
Urine-derived stem cells (USCs) are derived from urine and harbor the potential of proliferation and multidirectional differentiation. Moreover, USCs could be reprogrammed into pluripotent stem cells [namely urine-derived induced pluripotent stem cells (UiPSCs)] through transcription factors, such as octamer binding transcription factor 4, sex determining region Y-box 2, kruppel-like factor 4, myelocytomatosis oncogene, and Nanog homeobox and protein lin-28, in which the first four are known as Yamanaka factors. Mounting evidence supports that USCs and UiPSCs possess high potential of neurogenic, myogenic, and osteogenic differentiation, indicating that they may play a crucial role in the treatment of neurological and musculoskeletal diseases. Therefore, we summarized the origin and physiological characteristics of USCs and UiPSCs and their therapeutic application in neurological and musculoskeletal disorders in this review, which not only contributes to deepen our understanding of hallmarks of USCs and UiPSCs but also provides the theoretical basis for the treatment of neurological and musculoskeletal disorders with USCs and UiPSCs.
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Affiliation(s)
- Hui-Si Yang
- Department of Neurology and National Traditional Chinese Medicine Clinical Research Base, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
| | - Yue-Xiang Zheng
- Department of Neurology and National Traditional Chinese Medicine Clinical Research Base, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
| | - Xue Bai
- Department of Neurology and National Traditional Chinese Medicine Clinical Research Base, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
| | - Xiu-Ying He
- Department of Anesthesiology, Institute of Neurological Disease, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
| | - Ting-Hua Wang
- Department of Neurology and National Traditional Chinese Medicine Clinical Research Base, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
- Department of Anesthesiology, Institute of Neurological Disease, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
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Guo T, Wang J, Pang M, Liu W, Zhang X, Fan A, Liu H, Liu Q, Wei T, Li C, Zhao X, Lu Y. Reprogramming and multi-lineage transdifferentiation attenuate the tumorigenicity of colorectal cancer cells. J Biol Chem 2024; 300:105534. [PMID: 38072050 PMCID: PMC10801221 DOI: 10.1016/j.jbc.2023.105534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 11/18/2023] [Accepted: 11/27/2023] [Indexed: 01/02/2024] Open
Abstract
Significant advances have been made in reprogramming various somatic cells into induced pluripotent stem cells (iPSCs) and in multi-lineage differentiation (transdifferentiation) into different tissues. These manipulable transdifferentiating techniques may be applied in cancer therapy. Limited works have been reported that cancer cell malignancy can be switched to benign phenotypes through reprogramming techniques. Here, we reported that two colorectal cancer (CRC) cell lines (DLD1, HT29) could be reprogrammed into iPSCs (D-iPSCs, H-iPSCs). D- and H-iPSCs showed reduced tumorigenesis. Furthermore, we successfully induced D- and H-iPSCs differentiation into terminally differentiated cell types such as cardiomyocyte, neuron, and adipocyte-like cells. Impressively, the differentiated cells exhibited further attenuated tumorigenesis in vitro and in vivo. RNA-Seq further indicated that epigenetic changes occurred after reprogramming and transdifferentiation that caused reduced tumorigenicity. Overall, our study indicated that CRC cells can be reprogrammed and further differentiated into terminally differentiated lineages with attenuation of their malignancy in vitro and in vivo. The current work sheds light on a potential multi-lineage differentiation therapeutic strategy for colorectal cancer.
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Affiliation(s)
- Tongtong Guo
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Juan Wang
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Maogui Pang
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China; Department of Gastroenterology, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Wanning Liu
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Xiaohui Zhang
- Department of Gastroenterology, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Ahui Fan
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Hengtao Liu
- Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing, China
| | - Qianqian Liu
- Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing, China
| | - Tianying Wei
- Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing, China
| | - Cunxi Li
- Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing, China; Cytogenetics Laboratory, Beijing Institute of Human Genetics and Reproduction Medicine, Beijing, China.
| | - Xiaodi Zhao
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China.
| | - Yuanyuan Lu
- State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China.
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Astro V, Adamo A. Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies. Methods Mol Biol 2024; 2770:185-200. [PMID: 38351455 DOI: 10.1007/978-1-0716-3698-5_14] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/16/2024]
Abstract
Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.
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Affiliation(s)
- Veronica Astro
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
| | - Antonio Adamo
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
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Inagaki M. Cell Reprogramming and Differentiation Utilizing Messenger RNA for Regenerative Medicine. J Dev Biol 2023; 12:1. [PMID: 38535481 PMCID: PMC10971469 DOI: 10.3390/jdb12010001] [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: 10/30/2023] [Revised: 12/06/2023] [Accepted: 12/19/2023] [Indexed: 06/16/2024] Open
Abstract
The COVID-19 pandemic generated interest in the medicinal applications of messenger RNA (mRNA). It is expected that mRNA will be applied, not only to vaccines, but also to regenerative medicine. The purity of mRNA is important for its medicinal applications. However, the current mRNA synthesis techniques exhibit problems, including the contamination of undesired 5'-uncapped mRNA and double-stranded RNA. Recently, our group developed a completely capped mRNA synthesis technology that contributes to the progress of mRNA research. The introduction of chemically modified nucleosides, such as N1-methylpseudouridine and 5-methylcytidine, has been reported by Karikó and Weissman, opening a path for the practical application of mRNA for vaccines and regenerative medicine. Yamanaka reported the production of induced pluripotent stem cells (iPSCs) by introducing four types of genes using a retrovirus vector. iPSCs are widely used for research on regenerative medicine and the preparation of disease models to screen new drug candidates. Among the Yamanaka factors, Klf4 and c-Myc are oncogenes, and there is a risk of tumor development if these are integrated into genomic DNA. Therefore, regenerative medicine using mRNA, which poses no risk of genome insertion, has attracted attention. In this review, the author summarizes techniques for synthesizing mRNA and its application in regenerative medicine.
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Affiliation(s)
- Masahito Inagaki
- Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
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Guo T, Wei Q. Cell Reprogramming Techniques: Contributions to Cancer Therapy. Cell Reprogram 2023; 25:142-153. [PMID: 37530737 DOI: 10.1089/cell.2023.0011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/03/2023] Open
Abstract
The reprogramming of terminally differentiated cells over the past few years has become important for induced pluripotent stem cells (iPSCs) in the field of regenerative medicine and disease drug modeling. At the same time, iPSCs have also played an important role in human cancer research. iPSCs derived from cancer patients can be used to simulate the early progression of cancer, for drug testing, and to study the molecular mechanism of cancer occurrence. In recent years, with the application of cellular immunotherapy in cancer therapy, patient-derived iPSC-induced immune cells (T, natural killer, and macrophage cells) solve the problem of immune rejection and have higher immunogenicity, which greatly improves the therapeutic efficiency of immune cell therapy. With the continuous progress of cancer differentiation therapy, iPSC technology can reprogram cancer cells to a more primitive pluripotent undifferentiated state, and successfully reverse cancer cells to a benign phenotype by changing the epigenetic inheritance of cancer cells. This article reviews the recent progress of cell reprogramming technology in human cancer research, focuses on the application of reprogramming technology in cancer immunotherapy and the problems solved, and summarizes the malignant phenotype changes of cancer cells in the process of reprogramming and subsequent differentiation.
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Affiliation(s)
- Tongtong Guo
- College of Life Science, Northwest University, Xi'an, China
| | - Qi Wei
- Wuhan Institute of Technology, Wuhan, China
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Park S, Gwon Y, Khan SA, Jang KJ, Kim J. Engineering considerations of iPSC-based personalized medicine. Biomater Res 2023; 27:67. [PMID: 37420273 DOI: 10.1186/s40824-023-00382-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Accepted: 04/19/2023] [Indexed: 07/09/2023] Open
Abstract
Personalized medicine aims to provide tailored medical treatment that considers the clinical, genetic, and environmental characteristics of patients. iPSCs have attracted considerable attention in the field of personalized medicine; however, the inherent limitations of iPSCs prevent their widespread use in clinical applications. That is, it would be important to develop notable engineering strategies to overcome the current limitations of iPSCs. Such engineering approaches could lead to significant advances in iPSC-based personalized therapy by offering innovative solutions to existing challenges, from iPSC preparation to clinical applications. In this review, we summarize how engineering strategies have been used to advance iPSC-based personalized medicine by categorizing the development process into three distinctive steps: 1) the production of therapeutic iPSCs; 2) engineering of therapeutic iPSCs; and 3) clinical applications of engineered iPSCs. Specifically, we focus on engineering strategies and their implications for each step in the development of iPSC-based personalized medicine.
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Affiliation(s)
- Sangbae Park
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
- Institute of Nano-Stem Cells Therapeutics, NANOBIOSYSTEM Co, Ltd, Gwangju, 61011, Republic of Korea
| | - Yonghyun Gwon
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
| | - Shahidul Ahmed Khan
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
| | - Kyoung-Je Jang
- Department of Bio-Systems Engineering, Institute of Smart Farm, Gyeongsang National University, Jinju, 52828, Republic of Korea.
- Institute of Agriculture & Life Science, Gyeongsang National University, Jinju, 52828, Republic of Korea.
| | - Jangho Kim
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Institute of Nano-Stem Cells Therapeutics, NANOBIOSYSTEM Co, Ltd, Gwangju, 61011, Republic of Korea.
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Chenouard V, Leray I, Tesson L, Remy S, Allan A, Archer D, Caulder A, Fortun A, Bernardeau K, Cherifi Y, Teboul L, David L, Anegon I. Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions. iScience 2023; 26:106399. [PMID: 37034986 PMCID: PMC10074149 DOI: 10.1016/j.isci.2023.106399] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Revised: 01/03/2023] [Accepted: 03/09/2023] [Indexed: 03/16/2023] Open
Abstract
CRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 μM of Cas9, equimolar Cas9/gRNA ratio and 2 μM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models.
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Affiliation(s)
- Vanessa Chenouard
- INSERM, Nantes Université, CHU Nantes, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France
- genOway, Lyon 69007, France
| | - Isabelle Leray
- Nantes Université, CHU Nantes, Inserm, CNRS, BioCore, F-44000 Nantes, France
| | - Laurent Tesson
- INSERM, Nantes Université, CHU Nantes, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France
| | - Severine Remy
- INSERM, Nantes Université, CHU Nantes, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France
| | - Alasdair Allan
- Mary Lyon Centre, MRC Harwell Institute, Harwell Oxford, UK
| | - Daniel Archer
- Mary Lyon Centre, MRC Harwell Institute, Harwell Oxford, UK
| | - Adam Caulder
- Mary Lyon Centre, MRC Harwell Institute, Harwell Oxford, UK
| | - Agnès Fortun
- Nantes Université, CHU Nantes, CNRS, Inserm, BioCore, US16, Plateforme P2R, SFR Bonamy, F-44000 Nantes, France
- Cibles et Médicaments des Infections et du Cancer, IICiMed, Nantes Université, UR 1155, F-44000 Nantes, France
| | - Karine Bernardeau
- Nantes Université, CHU Nantes, CNRS, Inserm, BioCore, US16, Plateforme P2R, SFR Bonamy, F-44000 Nantes, France
| | | | - Lydia Teboul
- Mary Lyon Centre, MRC Harwell Institute, Harwell Oxford, UK
| | - Laurent David
- INSERM, Nantes Université, CHU Nantes, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France
- Nantes Université, CHU Nantes, Inserm, CNRS, BioCore, F-44000 Nantes, France
| | - Ignacio Anegon
- INSERM, Nantes Université, CHU Nantes, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France
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Maintenance of methylation profile in imprinting control regions in human induced pluripotent stem cells. Clin Epigenetics 2022; 14:190. [PMID: 36578048 PMCID: PMC9798676 DOI: 10.1186/s13148-022-01410-8] [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: 10/21/2022] [Accepted: 12/14/2022] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND Parental imprinting is an epigenetic mechanism that leads to monoallelic expression of a subset of genes depending on their parental origin. Imprinting disorders (IDs), caused by disturbances of imprinted genes, are a set of rare congenital diseases that mainly affect growth, metabolism and development. To date, there is no accurate model to study the physiopathology of IDs or test therapeutic strategies. Human induced pluripotent stem cells (iPSCs) are a promising cellular approach to model human diseases and complex genetic disorders. However, aberrant hypermethylation of imprinting control regions (ICRs) may appear during the reprogramming process and subsequent culture of iPSCs. Therefore, we tested various conditions of reprogramming and culture of iPSCs and performed an extensive analysis of methylation marks at the ICRs to develop a cellular model that can be used to study IDs. RESULTS We assessed the methylation levels at seven imprinted loci in iPSCs before differentiation, at various passages of cell culture, and during chondrogenic differentiation. Abnormal methylation levels were found, with hypermethylation at 11p15 H19/IGF2:IG-DMR and 14q32 MEG3/DLK1:IG-DMR, independently of the reprogramming method and cells of origin. Hypermethylation at these two loci led to the loss of parental imprinting (LOI), with biallelic expression of the imprinted genes IGF2 and DLK1, respectively. The epiPS™ culture medium combined with culturing of the cells under hypoxic conditions prevented hypermethylation at H19/IGF2:IG-DMR (ICR1) and MEG3/DLK1:IG-DMR, as well as at other imprinted loci, while preserving the proliferation and pluripotency qualities of these iPSCs. CONCLUSIONS An extensive and quantitative analysis of methylation levels of ICRs in iPSCs showed hypermethylation of certain ICRs in human iPSCs, especially paternally methylated ICRs, and subsequent LOI of certain imprinted genes. The epiPS™ culture medium and culturing of the cells under hypoxic conditions prevented hypermethylation of ICRs in iPSCs. We demonstrated that the reprogramming and culture in epiPS™ medium allow the generation of control iPSCs lines with a balanced methylation and ID patient iPSCs lines with unbalanced methylation. Human iPSCs are therefore a promising cellular model to study the physiopathology of IDs and test therapies in tissues of interest.
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Generation of CD34 +CD43 + Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells. Cells 2022; 11:cells11244046. [PMID: 36552810 PMCID: PMC9777438 DOI: 10.3390/cells11244046] [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: 10/19/2022] [Revised: 11/15/2022] [Accepted: 11/18/2022] [Indexed: 12/15/2022] Open
Abstract
Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.
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Long P, Shi Y, Sun F, Wei Y, Wu B, Li Q, Jie Q, Ma Y. Establishment of a non‐integrated induced pluripotent stem cell line derived from human chorionic villi cells. J Clin Lab Anal 2022; 36:e24464. [PMID: 35527669 PMCID: PMC9169189 DOI: 10.1002/jcla.24464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Revised: 04/17/2022] [Accepted: 04/18/2022] [Indexed: 11/17/2022] Open
Abstract
Background Few studies have investigated the generation of induced pluripotent stem cells (iPSCs) derived from human primary chorionic villi (CV) cells. The present study aimed to explore an optimal electroporation (EP) condition for generating non‐integrated iPSCs from CV cells (CV‐iPSCs). Methods The EGFP plasmid was transfected into CV cells under different EP conditions to evaluate cell adherence and the rate of EGFP positive cells. Subsequently, CV cells were transfected with the pEP4‐E02S‐ET2K and pCEP4‐miR‐302–367 plasmids under optimal EP conditions. Finally, CV‐iPSC pluripotency, karyotype analysis, and differentiation ability were investigated. Results Following EP for 48 h under different conditions, different confluency, and transfection efficiency were observed in CV cells. Higher cell density was observed in CV cells exposed to 200 V for 100 s, while higher transfection efficiency was obtained in cells electroporated at a pulse of 300 V for 300 s. To generate typical primitive iPSCs, CV cells were transfected with pEP4‐E02S‐ET2K and pCEP4‐miR‐302–367 plasmids using EP and were then cultured in induction medium for 20 days under selected conditions. Subsequently, monoclonal iPSCs were isolated and were evaluated pluripotency with AP positive staining, the expression of OCT4, SOX2, and NANOG in vitro and the formation of three germ layer teratomas in vivo. Conclusion CV‐iPSCs were successfully established under the conditions of 100 μl shock cup and EP pulse of 200 V for 300 s for two times. This may provide a novel strategy for investigating the pathogenesis of several diseases and gene therapy.
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Affiliation(s)
- Ping Long
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
- Guizhou Qiannan People's Hospital Guizhou China
| | - Yuechuan Shi
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
- Hainan Medical University Haikou Hainan China
| | - Fei Sun
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
- Department of Obstetrics and Gynecology of Nanfang Hospital Southern Medical University Guangzhou China
| | - Yunjian Wei
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
| | - Bangyong Wu
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
| | - Qi Li
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
| | - Qiuling Jie
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
| | - Yanlin Ma
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research The First Affiliated Hospital of Hainan Medical University Haikou Haikou Hainan China
- Key Laboratory of Tropical Translational Medicine of Ministry of Education Hainan Medical University Haikou Hainan China
- Haikou Key Laboratory of Human Genetic Resources Preservation of First Affiliated Hospital Hainan Medical University Haikou Hainan China
- Hainan Medical University Haikou Hainan China
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12
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Tan LS, Chen JT, Lim LY, Teo AKK. Manufacturing clinical-grade human induced pluripotent stem cell-derived beta cells for diabetes treatment. Cell Prolif 2022; 55:e13232. [PMID: 35474596 PMCID: PMC9357357 DOI: 10.1111/cpr.13232] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2021] [Revised: 01/26/2022] [Accepted: 03/28/2022] [Indexed: 12/25/2022] Open
Abstract
The unlimited proliferative capacity of human pluripotent stem cells (hPSCs) fortifies it as one of the most attractive sources for cell therapy application in diabetes. In the past two decades, vast research efforts have been invested in developing strategies to differentiate hPSCs into clinically suitable insulin‐producing endocrine cells or functional beta cells (β cells). With the end goal being clinical translation, it is critical for hPSCs and insulin‐producing β cells to be derived, handled, stored, maintained and expanded with clinical compliance. This review focuses on the key processes and guidelines for clinical translation of human induced pluripotent stem cell (hiPSC)‐derived β cells for diabetes cell therapy. Here, we discuss the (1) key considerations of manufacturing clinical‐grade hiPSCs, (2) scale‐up and differentiation of clinical‐grade hiPSCs into β cells in clinically compliant conditions and (3) mandatory quality control and product release criteria necessitated by various regulatory bodies to approve the use of the cell‐based products.
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Affiliation(s)
- Lay Shuen Tan
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore.,Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Precision Medicine Translational Research Programme (TRP), Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Juin Ting Chen
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore.,Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Precision Medicine Translational Research Programme (TRP), Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Lillian Yuxian Lim
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore
| | - Adrian Kee Keong Teo
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore.,Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Precision Medicine Translational Research Programme (TRP), Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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13
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Agrawal M, Rasiah PK, Bajwa A, Rajasingh J, Gangaraju R. Mesenchymal Stem Cell Induced Foxp3(+) Tregs Suppress Effector T Cells and Protect against Retinal Ischemic Injury. Cells 2021; 10:3006. [PMID: 34831229 PMCID: PMC8616393 DOI: 10.3390/cells10113006] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 10/22/2021] [Accepted: 10/29/2021] [Indexed: 12/02/2022] Open
Abstract
Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1β, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina's immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.
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Affiliation(s)
- Mona Agrawal
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA; (M.A.); (P.K.R.)
| | - Pratheepa Kumari Rasiah
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA; (M.A.); (P.K.R.)
| | - Amandeep Bajwa
- James D. Eason Transplant Institute, Department of Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA;
- Department of Genetics, Genomics, and Informatics, University of Tennessee Health Science Center, Memphis, TN 38163, USA
- Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA;
| | - Johnson Rajasingh
- Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA;
- Department of Bioscience Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Rajashekhar Gangaraju
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA; (M.A.); (P.K.R.)
- Department of Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
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14
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Towards Biohybrid Lung: Induced Pluripotent Stem Cell Derived Endothelial Cells as Clinically Relevant Cell Source for Biologization. MICROMACHINES 2021; 12:mi12080981. [PMID: 34442603 PMCID: PMC8401467 DOI: 10.3390/mi12080981] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 08/03/2021] [Accepted: 08/11/2021] [Indexed: 11/16/2022]
Abstract
In order to provide an alternative treatment option to lung transplantation for patients with end-stage lung disease, we aim for the development of an implantable biohybrid lung (BHL), based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Complete hemocompatibility of all blood contacting surfaces is crucial for long-lasting BHL durability and can be achieved by their endothelialization. Autologous endothelial cells (ECs) would be the ideal cell source, but their limited proliferation potential excludes them for this purpose. As induced pluripotent stem cell-derived ECs enable the generation of a large number of ECs, we assessed and compared their capacity to form a viable and confluent monolayer on HFM, while indicating physiologic EC-specific anti-thrombogenic and anti-inflammatory properties. ECs were generated from three different human iPSC lines, and seeded onto fibronectin-coated poly-4-methyl-1-pentene (PMP) HFM. Following phenotypical characterization, ECs were analyzed for their thrombogenic and inflammatory behavior with or without TNFα induction, using FACS and qRT-PCR. Complementary, leukocyte- and platelet adhesion assays were carried out. The capacity of the iPSC-ECs to reendothelialize cell-free monolayer areas was assessed in a scratch assay. ECs sourced from umbilical cord blood (hCBECs) were used as control. iPSC-derived ECs formed confluent monolayers on the HFM and showed the typical EC-phenotype by expression of VE-cadherin and collagen-IV. A low protein and gene expression level of E-selectin and tissue factor was detected for all iPSC-ECs and the hCBECs, while a strong upregulation of these markers was noted upon stimulation with TNFα. This was in line with the physiological and strong induction of leukocyte adhesion detected after treatment with TNFα, iPSC-EC and hCBEC monolayers were capable of reducing thrombocyte adhesion and repopulating scratched areas. iPSCs offer the possibility to provide patient-specific ECs in abundant numbers needed to cover all blood contacting surfaces of the BHL with a viable, non-thrombogenic and non-inflammatory monolayer. iPSC-EC clones can differ in terms of their reendothelialization rate, and pro-inflammatory response. However, a less profound inflammatory response may even be advantageous for BHL application. With the proven ability of the seeded iPSC-ECs to reduce thrombocyte adhesion, we expect that thrombotic events that could lead to BHL occlusion can be avoided, and thus, justifies further studies on enabling BHL long-term application.
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15
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Ray A, Joshi JM, Sundaravadivelu PK, Raina K, Lenka N, Kaveeshwar V, Thummer RP. An Overview on Promising Somatic Cell Sources Utilized for the Efficient Generation of Induced Pluripotent Stem Cells. Stem Cell Rev Rep 2021; 17:1954-1974. [PMID: 34100193 DOI: 10.1007/s12015-021-10200-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/02/2021] [Indexed: 01/19/2023]
Abstract
Human induced Pluripotent Stem Cells (iPSCs) have enormous potential in understanding developmental biology, disease modeling, drug discovery, and regenerative medicine. The initial human iPSC studies used fibroblasts as a starting cell source to reprogram them; however, it has been identified to be a less appealing somatic cell source by numerous studies due to various reasons. One of the important criteria to achieve efficient reprogramming is determining an appropriate starting somatic cell type to induce pluripotency since the cellular source has a major influence on the reprogramming efficiency, kinetics, and quality of iPSCs. Therefore, numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. This review provides an overview of promising easily accessible somatic cell sources isolated in non-invasive or minimally invasive manner such as keratinocytes, urine cells, and peripheral blood mononuclear cells used for the generation of human iPSCs derived from healthy and diseased subjects. Notably, iPSCs generated from one of these cell types derived from the patient will offer ethical and clinical advantages. In addition, these promising somatic cell sources have the potential to efficiently generate bona fide iPSCs with improved reprogramming efficiency and faster kinetics. This knowledge will help in establishing strategies for safe and efficient reprogramming and the generation of patient-specific iPSCs from these cell types.
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Affiliation(s)
- Arnab Ray
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Jahnavy Madhukar Joshi
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, 580009, Karnataka, India
| | - Pradeep Kumar Sundaravadivelu
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Khyati Raina
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
| | - Nibedita Lenka
- National Centre for Cell Science, S. P. Pune University Campus, Pune - 411007, Ganeshkhind, Maharashtra, India
| | - Vishwas Kaveeshwar
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, 580009, Karnataka, India.
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
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16
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A non-invasive method to generate induced pluripotent stem cells from primate urine. Sci Rep 2021; 11:3516. [PMID: 33568724 PMCID: PMC7876031 DOI: 10.1038/s41598-021-82883-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 01/19/2021] [Indexed: 01/30/2023] Open
Abstract
Comparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.
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17
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Jamal M, Bashir A, Al-Sayegh M, Huang GTJ. Oral tissues as sources for induced pluripotent stem cell derivation and their applications for neural, craniofacial, and dental tissue regeneration. CELL SOURCES FOR IPSCS 2021:71-106. [DOI: 10.1016/b978-0-12-822135-8.00007-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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18
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Dubois F, Gaignerie A, Flippe L, Heslan JM, Tesson L, Chesneau M, Haspot F, Conchon S, David L, Brouard S. Toward a better definition of hematopoietic progenitors suitable for B cell differentiation. PLoS One 2020; 15:e0243769. [PMID: 33320872 PMCID: PMC7737978 DOI: 10.1371/journal.pone.0243769] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 11/29/2020] [Indexed: 01/10/2023] Open
Abstract
The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34+CD43+ hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.
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Affiliation(s)
- Florian Dubois
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Anne Gaignerie
- Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
| | - Léa Flippe
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Jean-Marie Heslan
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
- Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
| | - Laurent Tesson
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Mélanie Chesneau
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Fabienne Haspot
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Sophie Conchon
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
| | - Laurent David
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
- Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
| | - Sophie Brouard
- Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France
- * E-mail:
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19
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Flippe L, Gaignerie A, Sérazin C, Baron O, Saulquin X, Themeli M, Guillonneau C, David L. Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells. Front Cell Dev Biol 2020; 8:577464. [PMID: 33195214 PMCID: PMC7606846 DOI: 10.3389/fcell.2020.577464] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 09/18/2020] [Indexed: 11/25/2022] Open
Abstract
Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.
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Affiliation(s)
- Léa Flippe
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Anne Gaignerie
- Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
| | - Céline Sérazin
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Olivier Baron
- Department of Pediatric Cardiac Surgery, University Hospital of Nantes, Nantes, France
| | | | - Maria Themeli
- Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Center (UMC), Amsterdam, Netherlands
| | - Carole Guillonneau
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France
| | - Laurent David
- Université de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.,LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France.,Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, FED 4203, Inserm UMS 016, CNRS UMS 3556, Nantes, France
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20
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Burdeyron P, Giraud S, Hauet T, Steichen C. Urine-derived stem/progenitor cells: A focus on their characterization and potential. World J Stem Cells 2020; 12:1080-1096. [PMID: 33178393 PMCID: PMC7596444 DOI: 10.4252/wjsc.v12.i10.1080] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 06/26/2020] [Accepted: 08/24/2020] [Indexed: 02/06/2023] Open
Abstract
Cell therapy, i.e., the use of cells to repair an affected tissue or organ, is at the forefront of regenerative and personalized medicine. Among the multiple cell types that have been used for this purpose [including adult stem cells such as mesenchymal stem cells or pluripotent stem cells], urine-derived stem cells (USCs) have aroused interest in the past years. USCs display classical features of mesenchymal stem cells such as differentiation capacity and immunomodulation. Importantly, they have the main advantage of being isolable from one sample of voided urine with a cheap and unpainful procedure, which is broadly applicable, whereas most adult stem cell types require invasive procedure. Moreover, USCs can be differentiated into renal cell types. This is of high interest for renal cell therapy-based regenerative approaches. This review will firstly describe the isolation and characterization of USCs. We will specifically present USC phenotype, which is not an object of consensus in the literature, as well as detail their differentiation capacity. In the second part of this review, we will present and discuss the main applications of USCs. These include use as a substrate to generate human induced pluripotent stem cells, but we will deeply focus on the use of USCs for cell therapy approaches with a detailed analysis depending on the targeted organ or system. Importantly, we will also focus on the applications that rely on the use of USC-derived products such as microvesicles including exosomes, which is a strategy being increasingly employed. In the last section, we will discuss the remaining barriers and challenges in the field of USC-based regenerative medicine.
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Affiliation(s)
- Perrine Burdeyron
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers 86021, France
- Faculté de Médecine et Pharmacie, Université de Poitiers, Poitiers 86021, France
| | - Sébastien Giraud
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers 86021, France
- Service de Biochimie, CHU de Poitiers, Poitiers 86021, France
| | - Thierry Hauet
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers 86021, France
- Faculté de Médecine et Pharmacie, Université de Poitiers, Poitiers 86021, France
- Service de Biochimie, CHU de Poitiers, Poitiers 86021, France
| | - Clara Steichen
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers 86021, France
- Faculté de Médecine et Pharmacie, Université de Poitiers, Poitiers 86021, France.
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21
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Bouma MJ, Arendzen CH, Mummery CL, Mikkers H, Freund C. Reprogramming Urine-Derived Cells using Commercially Available Self-Replicative RNA and a Single Electroporation. ACTA ACUST UNITED AC 2020; 55:e124. [PMID: 32956580 PMCID: PMC7540473 DOI: 10.1002/cpsc.124] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
We describe a protocol for efficient generation of human‐induced pluripotent stem cells (hiPSCs) from urine‐derived cells (UDCs) obtained from adult donors using self‐replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c‐MYC (ReproRNA‐OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4‐expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC‐derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA‐OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step‐by‐step description for generating clonal hiPSCs from UDCs using ReproRNA‐OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors. Basic Protocol: Reprogramming of urine‐derived cells using ReproRNA‐OKSGM Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry Support Protocol 2: Characterization of functional pluripotency of hiPSCs
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Affiliation(s)
- Marga J Bouma
- LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.,Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
| | - Christiaan H Arendzen
- LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.,Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
| | - Christine L Mummery
- LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.,Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
| | - Harald Mikkers
- LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.,Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Christian Freund
- LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.,Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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22
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Loffet E, Brossard L, Mahe MM. Pluripotent stem cell derived intestinal organoids with an enteric nervous system. Methods Cell Biol 2020; 159:175-199. [PMID: 32586442 DOI: 10.1016/bs.mcb.2020.04.012] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The use of human pluripotent stem cells (hPSCs) and differentiation techniques offer new ways to generate specific tissue. It is now possible to differentiate hPSC into human intestinal organoids that include an enteric nervous system. Using step-wise differentiation processes, we generate innervated intestinal organoids that form three-dimensional structures bearing an epithelium, neurons and glial cells embedded in a supporting mesenchyme. Innervated organoids further develop to a complex structure with similar organization and cellular differentiation as the developing intestine. These tools open up new fields of application in the study of the development and pathophysiology of enteric neuropathies. Herein, we describe the generation of both human intestinal organoids and vagal neural crest cells from hPSC and their combination into an innervated organoid. We also discuss technical considerations for these experiments, and highlight advantages and limitations of the system.
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Affiliation(s)
- Elise Loffet
- Université de Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France
| | - Lisa Brossard
- Université de Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France
| | - Maxime M Mahe
- Université de Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France; Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States; Department of Pediatrics, University of Cincinnati, Cincinnati, OH, United States.
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23
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Lanznaster D, Veyrat-Durebex C, Vourc’h P, Andres CR, Blasco H, Corcia P. Metabolomics: A Tool to Understand the Impact of Genetic Mutations in Amyotrophic Lateral Sclerosis. Genes (Basel) 2020; 11:genes11050537. [PMID: 32403313 PMCID: PMC7288444 DOI: 10.3390/genes11050537] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 04/27/2020] [Accepted: 04/27/2020] [Indexed: 12/12/2022] Open
Abstract
Metabolomics studies performed in patients with amyotrophic lateral sclerosis (ALS) reveal a set of distinct metabolites that can shed light on the pathological alterations taking place in each individual. Metabolites levels are influenced by disease status, and genetics play an important role both in familial and sporadic ALS cases. Metabolomics analysis helps to unravel the differential impact of the most common ALS-linked genetic mutations (as C9ORF72, SOD1, TARDBP, and FUS) in specific signaling pathways. Further, studies performed in genetic models of ALS reinforce the role of TDP-43 pathology in the vast majority of ALS cases. Studies performed in differentiated cells from ALS-iPSC (induced Pluripotent Stem Cells) reveal alterations in the cell metabolism that are also found in ALS models and ultimately in ALS patients. The development of metabolomics approaches in iPSC derived from ALS patients allow addressing and ultimately understanding the pathological mechanisms taking place in any patient. Lately, the creation of a "patient in a dish" will help to identify patients that may benefit from specific treatments and allow the implementation of personalized medicine.
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Affiliation(s)
- Débora Lanznaster
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- Correspondence:
| | - Charlotte Veyrat-Durebex
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- CHU de Tours, Service de Biochimie et Biologie Moléculaire, 37000 Tours, France
| | - Patrick Vourc’h
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- CHU de Tours, Service de Biochimie et Biologie Moléculaire, 37000 Tours, France
| | - Christian R. Andres
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- CHU de Tours, Service de Biochimie et Biologie Moléculaire, 37000 Tours, France
| | - Hélène Blasco
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- CHU de Tours, Service de Biochimie et Biologie Moléculaire, 37000 Tours, France
| | - Philippe Corcia
- UMR 1253, iBrain, University of Tours, Inserm, 37000 Tours, France; (C.V.-D.); (P.V.); (C.R.A.); (H.B.); (P.C.)
- CHU de Tours, Service de Neurologie, 37000 Tours, France
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24
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Urine-Derived Induced Pluripotent Stem Cells in Cardiovascular Disease. Cardiol Res Pract 2020; 2020:3563519. [PMID: 32377426 PMCID: PMC7199581 DOI: 10.1155/2020/3563519] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Revised: 11/26/2019] [Accepted: 12/17/2019] [Indexed: 12/28/2022] Open
Abstract
Recent studies have demonstrated that stem cells are equipped with the potential to differentiate into various types of cells, including cardiomyocytes. Meanwhile, stem cells are highly promising in curing cardiovascular diseases. However, owing to the ethical challenges posed in stem cell acquisition and the complexity and invasive nature of the method, large-scale expansions and clinical applications in the laboratory have been limited. The current generation of cardiomyocytes is available from diverse sources; urine is one of the promising sources among them. Although advanced research was established in the generation of human urine cells as cardiomyocytes, the reprogramming of urine cells to cardiomyocytes remains unclear. In this context, it is necessary to develop a minimally invasive method to create induced pluripotent stem cells (iPSCs). This review focuses on the latest advances in research on urine-derived iPSCs and their application mechanisms in cardiovascular diseases.
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25
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Urine-Derived Stem Cells: Applications in Regenerative and Predictive Medicine. Cells 2020; 9:cells9030573. [PMID: 32121221 PMCID: PMC7140531 DOI: 10.3390/cells9030573] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2019] [Revised: 02/17/2020] [Accepted: 02/25/2020] [Indexed: 12/14/2022] Open
Abstract
Despite being a biological waste, human urine contains a small population of cells with self-renewal capacity and differentiation potential into several cell types. Being derived from the convoluted tubules of nephron, renal pelvis, ureters, bladder and urethra, urine-derived stem cells (UDSC) have a similar phenotype to mesenchymal stroma cells (MSC) and can be reprogrammed into iPSC (induced pluripotent stem cells). Having simple, safer, low-cost and noninvasive collection procedures, the interest in UDSC has been growing in the last decade. With great potential in regenerative medicine applications, UDSC can also be used as biological models for pharmacology and toxicology tests. This review describes UDSC biological characteristics and differentiation potential and their possible use, including the potential of UDSC-derived iPSC to be used in drug discovery and toxicology, as well as in regenerative medicine. Being a new cellular platform amenable to noninvasive collection for disease stratification and personalized therapy could be a future application for UDSC.
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26
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NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate. Cells 2020; 9:cells9020509. [PMID: 32102328 PMCID: PMC7072849 DOI: 10.3390/cells9020509] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 12/11/2022] Open
Abstract
The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.
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27
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Sato M, Takizawa H, Nakamura A, Turner BJ, Shabanpoor F, Aoki Y. Application of Urine-Derived Stem Cells to Cellular Modeling in Neuromuscular and Neurodegenerative Diseases. Front Mol Neurosci 2019; 12:297. [PMID: 31920531 PMCID: PMC6915080 DOI: 10.3389/fnmol.2019.00297] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2019] [Accepted: 11/20/2019] [Indexed: 12/11/2022] Open
Abstract
Neuromuscular and neurodegenerative diseases are mostly modeled using genetically modified animals such as mice. However, animal models do not recapitulate all the phenotypes that are specific to human disease. This is mainly due to the genetic, anatomical and physiological difference in the neuromuscular systems of animals and humans. The emergence of direct and indirect human somatic cell reprogramming technologies may overcome this limitation because they enable the use of disease and patient-specific cellular models as enhanced platforms for drug discovery and autologous cell-based therapy. Induced pluripotent stem cells (iPSCs) and urine-derived stem cells (USCs) are increasingly employed to recapitulate the pathophysiology of various human diseases. Recent cell-based modeling approaches utilize highly complex differentiation systems that faithfully mimic human tissue- and organ-level dysfunctions. In this review, we discuss promising cellular models, such as USC- and iPSC-based approaches, that are currently being used to model human neuromuscular and neurodegenerative diseases.
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Affiliation(s)
- Mitsuto Sato
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.,Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan
| | - Hotake Takizawa
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan
| | - Akinori Nakamura
- Department of Clinical Research, National Hospital Organization Matsumoto Medical Center, Matsumoto, Japan
| | - Bradley J Turner
- The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia
| | - Fazel Shabanpoor
- The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia
| | - Yoshitsugu Aoki
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan
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28
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Doss MX, Sachinidis A. Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications. Cells 2019; 8:cells8050403. [PMID: 31052294 PMCID: PMC6562607 DOI: 10.3390/cells8050403] [Citation(s) in RCA: 271] [Impact Index Per Article: 45.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2019] [Revised: 04/23/2019] [Accepted: 04/26/2019] [Indexed: 12/17/2022] Open
Abstract
Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput "omics" methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy.
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Affiliation(s)
- Michael Xavier Doss
- Technology Development Division, BioMarin Pharmaceutical Inc, 105 Digital Drive, Novato, CA 94949, USA.
| | - Agapios Sachinidis
- Institute of Neurophysiology and Center for Molecular Medicine, University of Cologne, Robert-Koch Str. 39, 50931 Cologne, Germany.
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29
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Warren L, Lin C. mRNA-Based Genetic Reprogramming. Mol Ther 2019; 27:729-734. [PMID: 30598301 PMCID: PMC6453511 DOI: 10.1016/j.ymthe.2018.12.009] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Revised: 12/07/2018] [Accepted: 12/08/2018] [Indexed: 01/12/2023] Open
Abstract
The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient's own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several "non-integrating" reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously "footprint-free," most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic.
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Affiliation(s)
- Luigi Warren
- Cellular Reprogramming, Inc., Pasadena, CA, USA.
| | - Cory Lin
- Cellular Reprogramming, Inc., Pasadena, CA, USA
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