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Skamagki M, Zhang C, Hacisuleyman E, Galleti G, Wu C, Vinagolu RK, Cha H, Ata D, Kim J, Weiskittel T, Diop M, Aung T, Del Latto M, Kim AS, Li Z, Miele M, Zhao R, Tang LH, Hendrickson RC, Romesser PB, Smith JJ, Giannakakou P, Darnell RB, Bott MJ, Li H, Kim K. Aging-dependent dysregulation of EXOSC2 is maintained in cancer as a dependency. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.04.04.647279. [PMID: 40236131 PMCID: PMC11996493 DOI: 10.1101/2025.04.04.647279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Reprogramming of aged donor tissue cells into induced pluripotent stem cells (A-iPSC) preserved the epigenetic memory of aged-donor tissue, defined as genomic instability and poor tissue differentiation in our previous study. The unbalanced expression of RNA exosome subunits affects the RNA degradation complex function and is associated with geriatric diseases including premature aging and cancer progression. We hypothesized that the age-dependent progressive subtle dysregulation of EXOSC2 (exosome component 2) causes the aging traits (abnormal cell cycle and poor tissue differentiation). We used embryonic stem cells as a tool to study EXOSC2 function as the aging trait epigenetic memory determined in A-iPSC because these aging traits could not be studied in senesced aged cells or immortalized cancer cells. We found that the regulatory subunit of PP2A phosphatase, PPP2R5E, is a key target of EXOSC2 and this regulation is preserved in stem cells and cancer.
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Zhang Q, He J, Zhu D, Chen Y, Fu M, Lu S, Qiu Y, Zhou G, Yang G, Jiang Z. Genetically modified organoids for tissue engineering and regenerative medicine. Adv Colloid Interface Sci 2025; 335:103337. [PMID: 39547125 DOI: 10.1016/j.cis.2024.103337] [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: 01/18/2024] [Revised: 07/23/2024] [Accepted: 11/07/2024] [Indexed: 11/17/2024]
Abstract
To date, genetically modified organoids are emerging as a promising 3D modeling tool aimed at solving genetically relevant clinical and biomedical problems for regenerative medicine and tissue engineering. As an optimal vehicle for gene delivery, genetically modified organoids can enhance or reduce the expression of target genes through virus and non-virus-based gene transfection methods to achieve tissue regeneration. Animal experiments and preclinical studies have demonstrated the beneficial role of genetically modified organoids in various aspects of organ regeneration, including thymus, lacrimal glands, brain, lung, kidney, photoreceptors, etc. Furthermore, the technology offers a potential treatment option for various diseases, such as Fabry disease, non-alcoholic steatohepatitis, and Lynch syndrome. Nevertheless, the uncertain safety of genetic modification, the risk of organoid application, and bionics of current genetically modified organoids are still challenging. This review summarizes the researches on genetically modified organoids in recent years, and describes the transfection methods and functions of genetically modified organoids, then introduced their applications at length. Also, the limitations and future development directions of genetically modified organoids are included.
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Affiliation(s)
- Qinmeng Zhang
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Jin He
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Danji Zhu
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Yunxuan Chen
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Mengdie Fu
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Shifan Lu
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Yuesheng Qiu
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Guodong Zhou
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China
| | - Guoli Yang
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China.
| | - Zhiwei Jiang
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310000, China.
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Shum C, Han SY, Thiruvahindrapuram B, Wang Z, de Rijke J, Zhang B, Sundberg M, Chen C, Buttermore ED, Makhortova N, Howe J, Sahin M, Scherer SW. Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/Cas-edited iPSCs. Comput Struct Biotechnol J 2024; 23:638-647. [PMID: 38283851 PMCID: PMC10819409 DOI: 10.1016/j.csbj.2023.12.036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 12/22/2023] [Accepted: 12/23/2023] [Indexed: 01/30/2024] Open
Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.
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Affiliation(s)
- Carole Shum
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
- Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Sang Yeon Han
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | | | - Zhuozhi Wang
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Jill de Rijke
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Benjamin Zhang
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
- Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Maria Sundberg
- Department of Neurology, FM Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Cidi Chen
- Human Neuron Core, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | | | - Nina Makhortova
- Human Neuron Core, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Jennifer Howe
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
- Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Mustafa Sahin
- Department of Neurology, FM Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
- Rosamund Stone Zander Translational Neuroscience Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Stephen W. Scherer
- The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
- Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
- Department of Molecular Genetics and McLaughlin Centre, University of Toronto, Toronto, ON M5S 1A8, Canada
- Lead contact
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Beltran AS. Novel Approaches to Studying SLC13A5 Disease. Metabolites 2024; 14:84. [PMID: 38392976 PMCID: PMC10890222 DOI: 10.3390/metabo14020084] [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: 11/18/2023] [Revised: 01/17/2024] [Accepted: 01/18/2024] [Indexed: 02/25/2024] Open
Abstract
The role of the sodium citrate transporter (NaCT) SLC13A5 is multifaceted and context-dependent. While aberrant dysfunction leads to neonatal epilepsy, its therapeutic inhibition protects against metabolic disease. Notably, insights regarding the cellular and molecular mechanisms underlying these phenomena are limited due to the intricacy and complexity of the latent human physiology, which is poorly captured by existing animal models. This review explores innovative technologies aimed at bridging such a knowledge gap. First, I provide an overview of SLC13A5 variants in the context of human disease and the specific cell types where the expression of the transporter has been observed. Next, I discuss current technologies for generating patient-specific induced pluripotent stem cells (iPSCs) and their inherent advantages and limitations, followed by a summary of the methods for differentiating iPSCs into neurons, hepatocytes, and organoids. Finally, I explore the relevance of these cellular models as platforms for delving into the intricate molecular and cellular mechanisms underlying SLC13A5-related disorders.
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Affiliation(s)
- Adriana S Beltran
- Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA
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Yu N, Qiu J, Li K, Luo Q, Liu X, Yang Y, Jiang Z, He S, He A, Chen S, Chen X, Li Y, Ge J, Zhuang J, Yu K. Comparison of DNA stability and its related genes of neurons derived from induced pluripotent stem cells and primary retinal neurons. Cell Biol Int 2022; 46:1625-1636. [PMID: 35771585 DOI: 10.1002/cbin.11837] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 04/05/2022] [Accepted: 05/28/2022] [Indexed: 11/12/2022]
Abstract
Maintaining DNA stability in induced pluripotent stem cells (iPSCs) and iPSCs-derived neurons is a challenge in their clinical application. In the present study, we compared DNA stability between primary retinal neurons and differentiated neurons. We found that the basal level of γ-H2AX phosphorylation, a specific marker of DNA breaks, was notably higher (~26-folds) in human iPSCs compared to iPSCs-derived neurons. However, iPSCs-derived neurons are more sensitive to UV treatment compared to primary rat retinal neurons (postnatal Day 1). UV treatment induced a significantly decreasing in the cell viability of iPSCs-derived neurons by ~76.1%, whereas ~20.8% in primary retinal neurons. After analyzing the expression levels of genes involved in DNA stability, such as Brca1, Ligase IV, Ku80, and Mre11, we found that Ku80 and its heterodimeric partner, Ku70 were positive in iPSCs-derived neurons. However, both Ku80 and Ku70 are not expressed in primary retinal neurons and cerebellar neurons. Similarly, both Ku80 and Ku70 are also expressed in 3D retinal organoids from human embryonic stem cells (ESCs), except for a few Map2-negative cells and the hyaloid vessels of mice E12.5 retinas. Hence, Ku80, and Ku70 are specifically expressed in stem cell-derived neurons. Moreover, using the Ku80 inhibitor Compound L, our data showed that Ku80 promotes the DNA stability and cell viability of iPSCs-derived neurons. Thus, our results demonstrated that iPSCs-, ESCs-derived neurons have specific characteristics of DNA stability. This study provides new insights into the neural differentiation of stem cells but might also warrant the future clinical application of stem cells in neurodegenerative diseases.
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Affiliation(s)
- Na Yu
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Jin Qiu
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Kaijing Li
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Qian Luo
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Xuan Liu
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Ying Yang
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zihua Jiang
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Shengyu He
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Anqi He
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Shuilian Chen
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Xi Chen
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Yan Li
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Jian Ge
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Jing Zhuang
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Keming Yu
- State Key Laboratory of Ophthalmology, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
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Rossbach B, Hariharan K, Mah N, Oh SJ, Volk HD, Reinke P, Kurtz A. Human iPSC-Derived Renal Cells Change Their Immunogenic Properties during Maturation: Implications for Regenerative Therapies. Cells 2022; 11:cells11081328. [PMID: 35456007 PMCID: PMC9032821 DOI: 10.3390/cells11081328] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/11/2022] [Accepted: 04/12/2022] [Indexed: 12/24/2022] Open
Abstract
The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.
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Affiliation(s)
- Bella Rossbach
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
| | - Krithika Hariharan
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer Project Center for Stem Cell Processing, 97082 Würzburg, Germany
| | - Nancy Mah
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
| | - Su-Jun Oh
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
| | - Hans-Dieter Volk
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Institute for Medical Immunology (IMI), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Petra Reinke
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Berlin Center for Advanced Therapies (BeCat), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Andreas Kurtz
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
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Poetsch MS, Strano A, Guan K. Human induced pluripotent stem cells: From cell origin, genomic stability and epigenetic memory to translational medicine. Stem Cells 2022; 40:546-555. [PMID: 35291013 PMCID: PMC9216482 DOI: 10.1093/stmcls/sxac020] [Citation(s) in RCA: 60] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 03/06/2022] [Indexed: 11/14/2022]
Abstract
The potential of human induced pluripotent stem cells (iPSCs) to self-renew indefinitely and to differentiate virtually into any cell type in unlimited quantities makes them attractive for in-vitro disease modeling, drug screening, personalized medicine, and regenerative therapies. As the genome of iPSCs thoroughly reproduces that of the somatic cells from which they are derived, they may possess genetic abnormalities, which would seriously compromise their utility and safety. Genetic aberrations could be present in donor somatic cells and then transferred during iPSC generation, or they could occur as de novo mutations during reprogramming or prolonged cell culture. Therefore, to warrant safety of human iPSCs for clinical applications, analysis of genetic integrity, particularly during iPSC generation and differentiation, should be carried out on a regular basis. On the other hand, reprogramming of somatic cells to iPSCs requires profound modifications in the epigenetic landscape. Changes in chromatin structure by DNA methylations and histone tail modifications aim to reset the gene expression pattern of somatic cells to facilitate and establish self-renewal and pluripotency. However, residual epigenetic memory influences the iPSC phenotype, which may affect their application in disease therapeutics. The present review discusses the somatic cell origin, genetic stability, and epigenetic memory of iPSCs and their impact on basic and translational research.
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Affiliation(s)
- Mareike S Poetsch
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
| | - Anna Strano
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
| | - Kaomei Guan
- Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
- Corresponding author: Kaomei Guan, Institute of Pharmacology and Toxicology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany. Tel: +49 351 458 6246; Fax: +49 351 458 6315;
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8
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Choudhury S, Surendran N, Das A. Recent advances in the induced pluripotent stem cell-based skin regeneration. Wound Repair Regen 2021; 29:697-710. [PMID: 33970525 DOI: 10.1111/wrr.12925] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 03/30/2021] [Accepted: 04/27/2021] [Indexed: 01/05/2023]
Abstract
Skin regeneration has been a challenging clinical problem especially in cases of chronic wounds such as diabetic foot ulcers, and epidermolysis bullosa-related skin blisters. Prolonged non-healing wounds often lead to bacterial infections increasing the severity of wounds. Current treatment strategies for chronic wounds include debridement of wounds along with antibiotics, growth factors, and stem cell transplantation therapies. However, the compromised nature of autologous stem cells in patients with comorbidities such as diabetes limits the efficacy of the therapy. The discovery of induced pluripotent stem cell (iPSC) technology has immensely influenced the field of regenerative therapy. Enormous efforts have been made to develop integration-free iPSCs suitable for clinical therapies. This review focuses on recent advances in the methods and reprogramming factors for generating iPSCs along with the existing challenges such as genetic alterations, tumorigenicity, immune rejection, and regulatory hurdles for the clinical application of iPSCs. Furthermore, this review also highlights the benefits of using iPSCs for the generation of skin cells and skin disease modeling over the existing clinical therapies for skin regeneration in chronic wounds and skin diseases.
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Affiliation(s)
- Subholakshmi Choudhury
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
- Academy of Science and Innovative Research (AcSIR), Ghaziabad, India
| | - Nidhi Surendran
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
| | - Amitava Das
- Department of Applied Biology, Council of Scientific & Industrial Research-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India
- Academy of Science and Innovative Research (AcSIR), Ghaziabad, India
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9
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Zhang L, Pu K, Liu X, Bae SDW, Nguyen R, Bai S, Li Y, Qiao L. The Application of Induced Pluripotent Stem Cells Against Liver Diseases: An Update and a Review. Front Med (Lausanne) 2021; 8:644594. [PMID: 34277651 PMCID: PMC8280311 DOI: 10.3389/fmed.2021.644594] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Accepted: 06/04/2021] [Indexed: 11/13/2022] Open
Abstract
Liver diseases are a major health concern globally, and are associated with poor survival and prognosis of patients. This creates the need for patients to accept the main alternative treatment of liver transplantation to prevent progression to end-stage liver disease. Investigation of the molecular mechanisms underpinning complex liver diseases and their pathology is an emerging goal of stem cell scope. Human induced pluripotent stem cells (hiPSCs) derived from somatic cells are a promising alternative approach to the treatment of liver disease, and a prospective model for studying complex liver diseases. Here, we review hiPSC technology of cell reprogramming and differentiation, and discuss the potential application of hiPSC-derived liver cells, such as hepatocytes and cholangiocytes, in refractory liver-disease modeling and treatment, and drug screening and toxicity testing. We also consider hiPSC safety in clinical applications, based on genomic and epigenetic alterations, tumorigenicity, and immunogenicity.
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Affiliation(s)
- Lei Zhang
- The First Clinical Medical College, Lanzhou University, Lanzhou, China
- Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory of Biological Therapy and Regenerative Medicine Transformation Gansu Province, Lanzhou, China
| | - Ke Pu
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Xiaojun Liu
- Department of Medical Oncology, The First Hospital of Lanzhou University, Lanzhou, China
| | - Sarah Da Won Bae
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
| | - Romario Nguyen
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
| | - Suyang Bai
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Yi Li
- Department of Gastroenterology, The First Hospital of Lanzhou University, Lanzhou, China
- Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China
| | - Liang Qiao
- Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney at Westmead Clinical School, Westmead, NSW, Australia
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10
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Kosanke M, Osetek K, Haase A, Wiehlmann L, Davenport C, Schwarzer A, Adams F, Kleppa MJ, Schambach A, Merkert S, Wunderlich S, Menke S, Dorda M, Martin U. Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes. Mol Ther 2021; 29:2535-2553. [PMID: 33831558 DOI: 10.1016/j.ymthe.2021.04.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Revised: 03/03/2021] [Accepted: 04/02/2021] [Indexed: 02/06/2023] Open
Abstract
Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.
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Affiliation(s)
- Maike Kosanke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Katarzyna Osetek
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Alexandra Haase
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Lutz Wiehlmann
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Colin Davenport
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Adrian Schwarzer
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Felix Adams
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Marc-Jens Kleppa
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Axel Schambach
- Department of Hematology, Oncology and Stem Cell Transplantation, Institute of Experimental Hematology, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Sylvia Merkert
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Stephanie Wunderlich
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Sandra Menke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Marie Dorda
- Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany
| | - Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH - Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany.
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11
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Chromosomal aberration arises during somatic reprogramming to pluripotent stem cells. Cell Div 2020; 15:12. [PMID: 33292330 PMCID: PMC7641821 DOI: 10.1186/s13008-020-00068-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Accepted: 10/26/2020] [Indexed: 12/18/2022] Open
Abstract
Background Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) has opened new therapeutic possibilities. However, karyotypic abnormalities detected in iPSCs compromised their utility, especially chromosomal aberrations found at early passages raised serious safety concerns. The mechanism underlying the chromosomal abnormality in early-passage iPSCs is not known. Methods Human dermal fibroblasts (HDFs) were stimulated with KMOS (KLF4, cMYC, OCT4 and SOX2) proteins to enhance their proliferative capacity and many vigorous clones were obtained. Clonal reprogramming was carried out by KMOS mRNAs transfection to confirm the ‘chromosomal mutagenicity’ of reprogramming process. Subculturing was performed to examine karyotypic stability of iPSCs after the re-establishment of stemness. And antioxidant N-acetyl-cysteine (NAC) was added to the culture medium for further confirmming the mutagenicity in the first few days of reprogramming. Results Chromosomal aberrations were found in a small percentage of newly induced iPS clones by reprogramming transcription factors. Clonal reprogramming ruled out the aberrant chromosomes inherited from rare karyotypically abnormal parental cell subpopulation. More importantly, the antioxidant NAC effectively reduced the occurrence of chromosomal aberrations at the early stage of reprogramming. Once iPS cell lines were established, they restored karyotypic stability in subsequent subculturing. Conclusions Our results provided the first line of evidence for the ‘chromosomal mutagenicity’ of reprogramming process.
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Miao X, Li Y, Zheng C, Wang L, Jin C, Chen L, Mi S, Zhai W, Wang QF, Cai J. A promising iPS-based single-cell cloning strategy revealing signatures of somatic mutations in heterogeneous normal cells. Comput Struct Biotechnol J 2020; 18:2326-2335. [PMID: 32994891 PMCID: PMC7493045 DOI: 10.1016/j.csbj.2020.08.026] [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: 02/29/2020] [Revised: 08/15/2020] [Accepted: 08/26/2020] [Indexed: 11/24/2022] Open
Abstract
Single-cell genomics has advanced rapidly as trace-DNA amplification technologies evolved. However, current technologies are subject to a variety of pitfalls such as contamination, uneven genomic coverage, and amplification errors. Even for the "golden" strategy of single stem cell-derived clonal formation, high-fidelity amplification is applicable merely to single stem cells. It's still challenging to accurately define somatic mutations of a single cell in various cell types. Herein, we provided evidence, for the first time, to prove that induced pluripotent stem cells (iPS cells or iPSC), being a single somatic cell-derived clone, are recording almost identical (>90%) mutational profile of the initial cell progenitor. This finding demonstrates iPS technique, applicable to any cell type, can be utilized as a cell cloning strategy favorable for single-cell genomic amplification. This novel strategy is not limited by cell-type constraints or amplification artifacts, and thus enables our detailed investigation on the characteristics of somatic mutations in heterogeneous normal cells.
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Affiliation(s)
- Xuexia Miao
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
| | - Yueying Li
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Caihong Zheng
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
| | - Lifei Wang
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chen Jin
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lei Chen
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuangli Mi
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Weiwei Zhai
- Department of Human Genetics, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 138672, Singapore
- Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China
| | - Qian-Fei Wang
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jun Cai
- Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
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13
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Yoshihara M, Oguchi A, Murakawa Y. Genomic Instability of iPSCs and Challenges in Their Clinical Applications. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1201:23-47. [PMID: 31898780 DOI: 10.1007/978-3-030-31206-0_2] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Generation of human-induced pluripotent stem cells (iPSCs) from somatic cells has opened the possibility to design novel therapeutic approaches. In 2014, the first-in-human clinical trial of iPSC-based therapy was conducted. However, the transplantation for the second patient was discontinued at least in part due to genetic aberrations detected in iPSCs. Moreover, many studies have reported genetic aberrations in iPSCs with the rapid progress in genomic technologies. The presence of genomic instability raises serious safety concerns and can hamper the advancement of iPSC-based therapies. Here, we summarize our current knowledge on genomic instability of iPSCs and challenges in their clinical applications. In view of the recent expansion of stem cell therapies, it is crucial to gain deeper mechanistic insights into the genetic aberrations, ranging from chromosomal aberrations, copy number variations to point mutations. On the basis of their origin, these genetic aberrations in iPSCs can be classified as (i) preexisting mutations in parental somatic cells, (ii) reprogramming-induced mutations, and (iii) mutations that arise during in vitro culture. However, it is still unknown whether these genetic aberrations in iPSCs can be an actual risk factor for adverse effects. Intersection of the genomic data on iPSCs with the patients' clinical follow-up data will help to produce evidence-based criteria for clinical application. Furthermore, we discuss novel approaches to generate iPSCs with fewer genetic aberrations. Better understanding of iPSCs from both basic and clinical aspects will pave the way for iPSC-based therapies.
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Affiliation(s)
- Masahito Yoshihara
- Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
| | - Akiko Oguchi
- RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan
| | - Yasuhiro Murakawa
- RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan.
- IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy.
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14
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Perrera V, Martello G. How Does Reprogramming to Pluripotency Affect Genomic Imprinting? Front Cell Dev Biol 2019; 7:76. [PMID: 31143763 PMCID: PMC6521591 DOI: 10.3389/fcell.2019.00076] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Accepted: 04/18/2019] [Indexed: 01/14/2023] Open
Abstract
Human induced Pluripotent Stem Cells (hiPSCs) have the capacity to generate a wide range of somatic cells, thus representing an ideal tool for regenerative medicine. Patient-derived hiPSCs are also used for in vitro disease modeling and drug screenings. Several studies focused on the identification of DNA mutations generated, or selected, during the derivation of hiPSCs, some of which are known to drive cancer formation. Avoiding such stable genomic aberrations is paramount for successful use of hiPSCs, but it is equally important to ensure that their epigenetic information is correct, given the critical role of epigenetics in transcriptional regulation and its involvement in a plethora of pathologic conditions. In this review we will focus on genomic imprinting, a prototypical epigenetic mechanism whereby a gene is expressed in a parent-of-origin specific manner, thanks to the differential methylation of specific DNA sequences. Conventional hiPSCs are thought to be in a pluripotent state primed for differentiation. They display a hypermethylated genome with an unexpected loss of DNA methylation at imprinted loci. Several groups recently reported the generation of hiPSCs in a more primitive developmental stage, called naïve pluripotency. Naïve hiPSCs share several features with early human embryos, such as a global genome hypomethylation, which is also accompanied by a widespread loss of DNA methylation at imprinted loci. Given that loss of imprinting has been observed in genetic developmental disorders as well as in a wide range of cancers, it is fundamental to make sure that hiPSCs do not show such epigenetic aberrations. We will discuss what specific imprinted genes, associated with human pathologies, have been found commonly misregulated in hiPSCs and suggest strategies to effectively detect and avoid such undesirable epigenetic abnormalities.
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Affiliation(s)
- Valentina Perrera
- Department of Molecular Medicine, School of Medicine and Surgery, University of Padova, Padua, Italy
| | - Graziano Martello
- Department of Molecular Medicine, School of Medicine and Surgery, University of Padova, Padua, Italy
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15
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Simic MS, Moehle EA, Schinzel RT, Lorbeer FK, Halloran JJ, Heydari K, Sanchez M, Jullié D, Hockemeyer D, Dillin A. Transient activation of the UPR ER is an essential step in the acquisition of pluripotency during reprogramming. SCIENCE ADVANCES 2019; 5:eaaw0025. [PMID: 30989118 PMCID: PMC6457941 DOI: 10.1126/sciadv.aaw0025] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/08/2018] [Accepted: 02/25/2019] [Indexed: 05/11/2023]
Abstract
Somatic cells can be reprogrammed into pluripotent stem cells using the Yamanaka transcription factors. Reprogramming requires both epigenetic landscape reshaping and global remodeling of cell identity, structure, basic metabolic processes, and organelle form and function. We hypothesize that variable regulation of the proteostasis network and its influence upon the protein-folding environment within cells and their organelles is responsible for the low efficiency and stochasticity of reprogramming. We find that the unfolded protein response of the endoplasmic reticulum (UPRER), the mitochondrial UPR, and the heat shock response, which ensure proteome quality during stress, are activated during reprogramming. The UPRER is particularly crucial, and its ectopic, transient activation, genetically or pharmacologically, enhances reprogramming. Last, stochastic activation of the UPRER predicts reprogramming efficiency in naïve cells. Thus, the low efficiency and stochasticity of cellular reprogramming are due partly to the inability to properly initiate the UPRER to remodel the ER and its proteome.
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Affiliation(s)
- Milos S. Simic
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
| | - Erica A. Moehle
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
| | - Robert T. Schinzel
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
| | | | - Jonathan J. Halloran
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
| | | | - Melissa Sanchez
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
| | - Damien Jullié
- University of California, San Francisco, San Francisco, CA 94143, USA
| | | | - Andrew Dillin
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
- California Institute for Regenerative Medicine, Berkeley, CA 94720, USA
- University of California, Berkeley, Berkeley, CA 94720, USA
- Corresponding author.
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Attwood SW, Edel MJ. iPS-Cell Technology and the Problem of Genetic Instability-Can It Ever Be Safe for Clinical Use? J Clin Med 2019; 8:E288. [PMID: 30823421 PMCID: PMC6462964 DOI: 10.3390/jcm8030288] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 02/23/2019] [Accepted: 02/25/2019] [Indexed: 12/20/2022] Open
Abstract
The use of induced Pluripotent Stem Cells (iPSC) as a source of autologous tissues shows great promise in regenerative medicine. Nevertheless, several major challenges remain to be addressed before iPSC-derived cells can be used in therapy, and experience of their clinical use is extremely limited. In this review, the factors affecting the safe translation of iPSC to the clinic are considered, together with an account of efforts being made to overcome these issues. The review draws upon experiences with pluripotent stem-cell therapeutics, including clinical trials involving human embryonic stem cells and the widely transplanted mesenchymal stem cells. The discussion covers concerns relating to: (i) the reprogramming process; (ii) the detection and removal of incompletely differentiated and pluripotent cells from the resulting medicinal products; and (iii) genomic and epigenetic changes, and the evolutionary and selective processes occurring during culture expansion, associated with production of iPSC-therapeutics. In addition, (iv) methods for the practical culture-at-scale and standardization required for routine clinical use are considered. Finally, (v) the potential of iPSC in the treatment of human disease is evaluated in the light of what is known about the reprogramming process, the behavior of cells in culture, and the performance of iPSC in pre-clinical studies.
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Affiliation(s)
- Stephen W Attwood
- Department of Life Sciences, The Natural History Museum, London SW7 5BD, UK.
| | - Michael J Edel
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK.
- Control of Pluripotency Laboratory, Department of Physiological Sciences I, Faculty of Medicine, University of Barcelona, Hospital Clinic, Casanova 143, 08036 Barcelona, Spain.
- Victor Chang Cardiac Research Institute, Sydney, NSW 2145, Australia.
- Harry Perkins Research Institute, Fiona Stanley Hospital, University of Western Australia, PO Box 404, Bull Creek, Western Australia 6149, Australia.
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Mishra S, Kacin E, Stamatiadis P, Franck S, Van der Jeught M, Mertes H, Pennings G, De Sutter P, Sermon K, Heindryckx B, Geens M. The role of the reprogramming method and pluripotency state in gamete differentiation from patient-specific human pluripotent stem cells. Mol Hum Reprod 2019; 24:173-184. [PMID: 29471503 DOI: 10.1093/molehr/gay007] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Accepted: 02/10/2018] [Indexed: 12/16/2022] Open
Abstract
The derivation of gametes from patient-specific pluripotent stem cells may provide new perspectives for genetic parenthood for patients currently facing sterility. We use current data to assess the gamete differentiation potential of patient-specific pluripotent stem cells and to determine which reprogramming strategy holds the greatest promise for future clinical applications. First, we compare the two best established somatic cell reprogramming strategies: the production of induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer followed by embryonic stem cell derivation (SCNT-ESC). Recent reports have indicated that these stem cells, though displaying a similar pluripotency potential, show important differences at the epigenomic level, which may have repercussions on their applicability. By comparing data on the genetic and epigenetic stability of these cell types during derivation and in-vitro culture, we assess the reprogramming efficiency of both technologies and possible effects on the subsequent differentiation potential of these cells. Moreover, we discuss possible implications of mitochondrial heteroplasmy. We also address the ethical aspects of both cell types, as well as the safety considerations associated with clinical applications using these cells, e.g. the known genomic instability of human PSCs during long-term culture. Secondly, we discuss the role of the stem cell pluripotency state in germ cell differentiation. In mice, success in germ cell development from pluripotent stem cells could only be achieved when starting from a naive state of pluripotency. It remains to be investigated if the naive state is also crucial for germ cell differentiation in human cells and to what extent human naive pluripotency resembles the naive state in mouse.
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Affiliation(s)
- S Mishra
- Ghent-Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - E Kacin
- Research Group, Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Brussels, Belgium
| | - P Stamatiadis
- Ghent-Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - S Franck
- Research Group, Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Brussels, Belgium
| | - M Van der Jeught
- Ghent-Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - H Mertes
- Bioethics Institute Ghent, Department of Philosophy and Moral Sciences, Blandijnberg 2, 9000 Ghent, Belgium
| | - G Pennings
- Bioethics Institute Ghent, Department of Philosophy and Moral Sciences, Blandijnberg 2, 9000 Ghent, Belgium
| | - P De Sutter
- Ghent-Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - K Sermon
- Research Group, Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Brussels, Belgium
| | - B Heindryckx
- Ghent-Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - M Geens
- Research Group, Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Brussels, Belgium
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18
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Rashed AR, Gudulluoglu B, Yun HW, Habib M, Boyaci IH, Hong SH, Ozbay E, Caglayan H. Highly-Sensitive Refractive Index Sensing by Near-infrared Metatronic Nanocircuits. Sci Rep 2018; 8:11457. [PMID: 30061578 PMCID: PMC6065432 DOI: 10.1038/s41598-018-29623-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2018] [Accepted: 07/09/2018] [Indexed: 11/20/2022] Open
Abstract
In this work, we present a highly-sensitive refractive index sensor based on metatronic nanocircuits operating at near-infrared spectral range. The structure is designed based on simple nanorod geometry and fabricated by nanopatterning of transparent conducting oxides. The functionality of these polarization dependent metatronic nanocircuits is enhanced by applying tunable response. This feature is investigated by depositing NH2 (Amine) groups via plasma polymerization technique on top of indium-tin-oxide nanorods. The dielectric constant of Amine groups is a function of their thickness, which can be controlled by the RF power and the time duration of the applied plasma polymerization process. The resonance wavelengths of nanocircuits shift to higher wavelength, as the dielectric constant of the deposited material increases. An excellent agreement between the design and experimental results are obtained. Our metatronic based nanosensor offers a high-sensitive performance of 1587 nm/RIU with a satisfactory figure of merit for this class of sensors.
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Affiliation(s)
- A R Rashed
- Laboratory of Photonics, Tampere University of Technology, 33720, Tampere, Finland. .,Nanotechnology Research Center, Bilkent University, Bilkent, 06800, Ankara, Turkey.
| | - B Gudulluoglu
- Nanotechnology Research Center, Bilkent University, Bilkent, 06800, Ankara, Turkey.,Hacettepe University, Nanoscience and Nanomedicine Department, 06800, Ankara, Turkey
| | - H W Yun
- Components & Materials Research Laboratory, Electronics and Telecommunication Research Institute (ETRI), Daejeon, 305-350, Republic of Korea
| | - M Habib
- Nanotechnology Research Center, Bilkent University, Bilkent, 06800, Ankara, Turkey
| | - I H Boyaci
- Hacettepe University, Food Engineering, 06800, Ankara, Turkey
| | - S H Hong
- Components & Materials Research Laboratory, Electronics and Telecommunication Research Institute (ETRI), Daejeon, 305-350, Republic of Korea
| | - E Ozbay
- Nanotechnology Research Center, Bilkent University, Bilkent, 06800, Ankara, Turkey
| | - H Caglayan
- Laboratory of Photonics, Tampere University of Technology, 33720, Tampere, Finland.
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Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells. Cell Rep 2018; 21:308-315. [PMID: 29020618 DOI: 10.1016/j.celrep.2017.09.060] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2016] [Revised: 06/13/2017] [Accepted: 09/18/2017] [Indexed: 12/13/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) are generated by direct reprogramming of somatic cells and hold great promise for novel therapies. However, several studies have reported genetic variations in iPSC genomes. Here, we investigated point mutations identified by whole-genome sequencing in mouse and human iPSCs in the context of epigenetic status. In contrast to disease-causing single-nucleotide polymorphisms, de novo point mutations introduced during reprogramming were underrepresented in protein-coding genes and in open chromatin regions, including transcription factor binding sites. Instead, these mutations occurred preferentially in structurally condensed lamina-associated heterochromatic domains, suggesting that chromatin organization is a factor that can bias the regional mutation rate in iPSC genomes. Mutation signature analysis implicated oxidative stress associated with reprogramming as a likely cause of point mutations. Altogether, our study provides deeper understanding of the mutational landscape of iPSC genomes, paving an important way toward the translation of iPSC-based cell therapy.
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20
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Manipulating cell fate while confronting reproducibility concerns. Biochem Pharmacol 2018; 151:144-156. [DOI: 10.1016/j.bcp.2018.01.016] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 01/04/2018] [Indexed: 12/13/2022]
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21
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Hoffman GE, Hartley BJ, Flaherty E, Ladran I, Gochman P, Ruderfer DM, Stahl EA, Rapoport J, Sklar P, Brennand KJ. Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains. Nat Commun 2017; 8:2225. [PMID: 29263384 PMCID: PMC5738408 DOI: 10.1038/s41467-017-02330-5] [Citation(s) in RCA: 106] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2017] [Accepted: 11/20/2017] [Indexed: 12/20/2022] Open
Abstract
The power of human induced pluripotent stem cell (hiPSC)-based studies to resolve the smaller effects of common variants within the size of cohorts that can be realistically assembled remains uncertain. We identified and accounted for a variety of technical and biological sources of variation in a large case/control schizophrenia (SZ) hiPSC-derived cohort of neural progenitor cells and neurons. Reducing the stochastic effects of the differentiation process by correcting for cell type composition boosted the SZ signal and increased the concordance with post-mortem data sets. We predict a growing convergence between hiPSC and post-mortem studies as both approaches expand to larger cohort sizes. For studies of complex genetic disorders, to maximize the power of hiPSC cohorts currently feasible, in most cases and whenever possible, we recommend expanding the number of individuals even at the expense of the number of replicate hiPSC clones.
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Affiliation(s)
- Gabriel E Hoffman
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
- Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
| | - Brigham J Hartley
- Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Erin Flaherty
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Ian Ladran
- Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Peter Gochman
- Childhood Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Douglas M Ruderfer
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Division of Genetic Medicine, Departments of Medicine, Psychiatry and Biomedical Informatics, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, 37232, USA
| | - Eli A Stahl
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Judith Rapoport
- Childhood Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Pamela Sklar
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Kristen J Brennand
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
- Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
- Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
- Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
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22
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Preza E, Hardy J, Warner T, Wray S. Review: Induced pluripotent stem cell models of frontotemporal dementia. Neuropathol Appl Neurobiol 2017; 42:497-520. [PMID: 27291591 DOI: 10.1111/nan.12334] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2016] [Revised: 06/09/2016] [Accepted: 06/13/2016] [Indexed: 12/11/2022]
Abstract
The increasing prevalence of dementia in the ageing population combined with the lack of treatments and the burden on national health care systems globally make dementia a public health priority. Despite the plethora of important research findings published over the past two decades, the mechanisms underlying dementia are still poorly understood and the progress in pharmacological interventions is limited. Recent advances in cellular reprogramming and genome engineering technologies offer an unprecedented new paradigm in disease modeling. Induced pluripotent stem cells (iPSCs) have enabled the study of patient-derived neurons in vitro, a significant progress in the field of dementia research. The first studies using iPSCs to model dementia have recently emerged, holding promise for elucidating disease pathogenic mechanisms and accelerating drug discovery. In this review, we summarize the major findings of iPSC-based studies in frontotemporal dementia (FTD) and FTD overlapping with amyotrophic lateral sclerosis (FTD/ALS). We also discuss some of the main challenges in the use of iPSCs to model complex, late-onset neurodegenerative diseases such as dementias.
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Affiliation(s)
- E Preza
- Department of Molecular Neuroscience, UCL Institute of Neurology, London, WC1N 1PJ, UK.
| | - J Hardy
- Department of Molecular Neuroscience, UCL Institute of Neurology, London, WC1N 1PJ, UK
| | - T Warner
- Reta Lila Weston Institute of Neurological Studies, UCL Institute of Neurology, London, WC1N 1PJ, UK
| | - S Wray
- Department of Molecular Neuroscience, UCL Institute of Neurology, London, WC1N 1PJ, UK
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23
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Yoshihara M, Hayashizaki Y, Murakawa Y. Genomic Instability of iPSCs: Challenges Towards Their Clinical Applications. Stem Cell Rev Rep 2017; 13:7-16. [PMID: 27592701 PMCID: PMC5346115 DOI: 10.1007/s12015-016-9680-6] [Citation(s) in RCA: 196] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells generated directly from mature cells through the introduction of key transcription factors. iPSCs can be propagated and differentiated into many cell types in the human body, holding enormous potential in the field of regenerative medicine. However, genomic instability of iPSCs has been reported with the advent of high-throughput technologies such as next-generation sequencing. The presence of genetic variations in iPSCs has raised serious safety concerns, hampering the advancement of iPSC-based novel therapies. Here we summarize our current knowledge on genomic instability of iPSCs, with a particular focus on types of genetic variations and their origins. Importantly, it remains elusive whether genetic variations in iPSCs can be an actual risk factor for adverse effects including malignant outgrowth. Furthermore, we discuss novel approaches to generate iPSCs with fewer genetic variations. Lastly, we outline the safety issues and monitoring strategies of iPSCs in clinical settings.
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Affiliation(s)
- Masahito Yoshihara
- Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.,Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | | | - Yasuhiro Murakawa
- Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan. .,RIKEN Preventive Medicine and Diagnosis Innovation Program, Wako, Saitama, Japan.
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24
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Turinetto V, Orlando L, Giachino C. Induced Pluripotent Stem Cells: Advances in the Quest for Genetic Stability during Reprogramming Process. Int J Mol Sci 2017; 18:E1952. [PMID: 28902128 PMCID: PMC5618601 DOI: 10.3390/ijms18091952] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Revised: 09/01/2017] [Accepted: 09/08/2017] [Indexed: 12/19/2022] Open
Abstract
Evaluation of the extent and nature of induced pluripotent stem cell (iPSC) genetic instability is important for both basic research and future clinical use. As previously demonstrated regarding embryonic stem cells, such DNA aberrations might affect the differentiation capacity of the cells and increase their tumorigenicity. Here, we first focus on the contribution of multiple DNA damage response pathways during cellular reprogramming. We then discuss the origin and mechanisms responsible for the modification of genetic material in iPSCs (pre-existing variations in somatic cells, mutations induced by reprogramming factors, and mutations induced by culture expansion) and deepen the possible functional consequences of genetic variations in these cells. Lastly, we present some recent improvements of iPSC generation methods aimed at obtaining cells with fewer genetic variations.
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Affiliation(s)
- Valentina Turinetto
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Turin, Italy.
| | - Luca Orlando
- McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8S 4L8, Canada.
| | - Claudia Giachino
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Turin, Italy.
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25
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Römer C, Singh M, Hurst LD, Izsvák Z. How to tame an endogenous retrovirus: HERVH and the evolution of human pluripotency. Curr Opin Virol 2017; 25:49-58. [PMID: 28750248 DOI: 10.1016/j.coviro.2017.07.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2017] [Revised: 06/13/2017] [Accepted: 07/03/2017] [Indexed: 12/18/2022]
Abstract
HERVH is one of the most successful endogenous retrovirus in the human genome. Relative to other endogenous retroviruses, slower degradation of HERVH internal sequences indicates their potential relevance for the host. HERVH is transcriptionally active during human preimplantation embryogenesis. In this review, we focus on the role of HERVH in regulating human pluripotency. The HERVH-mediated pluripotency network has been evolved recently in primates. Nevertheless, it became an essential feature of human pluripotency. We discuss how HERVH modulates the human pluripotency network by providing alternative transcription factor binding sites, functioning as a long-range enhancer, and as being a major source for pluripotency specific long non-coding RNAs and chimeric transcripts.
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Affiliation(s)
- Christine Römer
- Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Manvendra Singh
- Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Laurence D Hurst
- Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Zsuzsanna Izsvák
- Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.
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26
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Ishikawa T. Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue. World J Stem Cells 2017; 9:77-88. [PMID: 28596815 PMCID: PMC5440771 DOI: 10.4252/wjsc.v9.i5.77] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 04/03/2017] [Accepted: 05/05/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing.
METHODS Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer (OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer.
RESULTS In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.
CONCLUSION The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.
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27
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Molchadsky A, Rotter V. p53 and its mutants on the slippery road from stemness to carcinogenesis. Carcinogenesis 2017; 38:347-358. [PMID: 28334334 DOI: 10.1093/carcin/bgw092] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2016] [Accepted: 08/25/2016] [Indexed: 12/18/2022] Open
Abstract
Normal development, tissue homeostasis and regeneration following injury rely on the proper functions of wide repertoire of stem cells (SCs) persisting during embryonic period and throughout the adult life. Therefore, SCs employ robust mechanisms to preserve their genomic integrity and avoid heritage of mutations to their daughter cells. Importantly, propagation of SCs with faulty DNA as well as dedifferentiation of genomically altered somatic cells may result in derivation of cancer SCs, which are considered to be the driving force of the tumorigenic process. Multiple experimental evidence suggest that p53, the central tumor suppressor gene, plays a critical regulatory role in determination of SCs destiny, thereby eliminating damaged SCs from the general SC population. Notably, mutant p53 proteins do not only lose the tumor suppressive function, but rather gain new oncogenic function that markedly promotes various aspects of carcinogenesis. In this review, we elaborate on the role of wild type and mutant p53 proteins in the various SCs types that appear under homeostatic conditions as well as in cancer. It is plausible that the growing understanding of the mechanisms underlying cancer SC phenotype and p53 malfunction will allow future optimization of cancer therapeutics in the context of precision medicine.
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Affiliation(s)
- Alina Molchadsky
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Varda Rotter
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
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28
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Panopoulos AD, D'Antonio M, Benaglio P, Williams R, Hashem SI, Schuldt BM, DeBoever C, Arias AD, Garcia M, Nelson BC, Harismendy O, Jakubosky DA, Donovan MKR, Greenwald WW, Farnam K, Cook M, Borja V, Miller CA, Grinstein JD, Drees F, Okubo J, Diffenderfer KE, Hishida Y, Modesto V, Dargitz CT, Feiring R, Zhao C, Aguirre A, McGarry TJ, Matsui H, Li H, Reyna J, Rao F, O'Connor DT, Yeo GW, Evans SM, Chi NC, Jepsen K, Nariai N, Müller FJ, Goldstein LSB, Izpisua Belmonte JC, Adler E, Loring JF, Berggren WT, D'Antonio-Chronowska A, Smith EN, Frazer KA. iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types. Stem Cell Reports 2017; 8:1086-1100. [PMID: 28410642 PMCID: PMC5390244 DOI: 10.1016/j.stemcr.2017.03.012] [Citation(s) in RCA: 120] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Revised: 03/08/2017] [Accepted: 03/13/2017] [Indexed: 11/18/2022] Open
Abstract
Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.
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Affiliation(s)
- Athanasia D Panopoulos
- Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Matteo D'Antonio
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Paola Benaglio
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
| | - Roy Williams
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA; Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Sherin I Hashem
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Bernhard M Schuldt
- Zentrum für Integrative Psychiatrie, Universitätsklinikum Schleswig-Holstein, 24105 Kiel, Germany
| | - Christopher DeBoever
- Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Angelo D Arias
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
| | - Melvin Garcia
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Bradley C Nelson
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Olivier Harismendy
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - David A Jakubosky
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Margaret K R Donovan
- Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - William W Greenwald
- Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - KathyJean Farnam
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Megan Cook
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Victor Borja
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Carl A Miller
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jonathan D Grinstein
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Frauke Drees
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jonathan Okubo
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | | | - Yuriko Hishida
- Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Veronica Modesto
- Stem Cell Core, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Carl T Dargitz
- Stem Cell Core, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Rachel Feiring
- Stem Cell Core, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Chang Zhao
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Aitor Aguirre
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Thomas J McGarry
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Hiroko Matsui
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - He Li
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Joaquin Reyna
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Fangwen Rao
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Daniel T O'Connor
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Gene W Yeo
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Sylvia M Evans
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Neil C Chi
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kristen Jepsen
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Naoki Nariai
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
| | - Franz-Josef Müller
- Zentrum für Integrative Psychiatrie, Universitätsklinikum Schleswig-Holstein, 24105 Kiel, Germany
| | - Lawrence S B Goldstein
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | | | - Eric Adler
- Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jeanne F Loring
- Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - W Travis Berggren
- Stem Cell Core, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | | | - Erin N Smith
- Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kelly A Frazer
- Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.
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29
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iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations. Proc Natl Acad Sci U S A 2017; 114:1964-1969. [PMID: 28167771 DOI: 10.1073/pnas.1616035114] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.
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30
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Abstract
Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. The advent of cell reprogramming technologies has widely disclosed unprecedented opportunities to generate renewable sources of human neural cells for disease modeling, drug discovery, and cell therapeutics. Both somatic reprogramming into induced pluripotent stem cells (iPSCs) and directly induced Neurons (iNeurons) rely on transcription factor-based cellular conversion processes. Nevertheless, they rely on very distinct mechanisms, biological barriers, technical limitations, different levels of efficiency, and generate neural cells with distinctive properties. Human iPSCs represent a long-term renewable source of neural cells, but over time genomic aberrations might erode the quality of the cultures and the in vitro differentiation process requires extensive time. Conversely, direct neuronal reprogramming ensures a fast and straightforward generation of iNeurons endowed with functional properties. However, in this last case, conversion efficiency is reduced when starting from adult human cells, and the molecular and functional fidelity of iNeurons with respect to their corresponding native neuronal subtype is yet to be fully ascertained in many cases. For any biomedical research application, it should be carefully pondered the reprogramming method to use for generating reprogrammed human neuronal subtypes that best fit with the following analysis considering the existing limitations and gap of knowledge still present in this young field of investigation.
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Affiliation(s)
- Vania Broccoli
- San Raffaele Scientific Institute, Milan, Italy; CNR-Institute of Neuroscience, Milan, Italy.
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31
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Gómez-Cabello D, Checa-Rodríguez C, Abad M, Serrano M, Huertas P. CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells. Stem Cell Reports 2017; 8:432-445. [PMID: 28065643 PMCID: PMC5311465 DOI: 10.1016/j.stemcr.2016.12.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2016] [Revised: 12/07/2016] [Accepted: 12/08/2016] [Indexed: 12/21/2022] Open
Abstract
Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs). All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.
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Affiliation(s)
- Daniel Gómez-Cabello
- Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville 41092, Spain.
| | - Cintia Checa-Rodríguez
- Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville 41092, Spain; Department of Genetics, University of Seville, Seville 41012, Spain
| | - María Abad
- Tumour Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Manuel Serrano
- Tumour Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Pablo Huertas
- Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville 41092, Spain; Department of Genetics, University of Seville, Seville 41012, Spain.
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32
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Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders. Sci Rep 2016; 6:26342. [PMID: 27197874 PMCID: PMC4873825 DOI: 10.1038/srep26342] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Accepted: 04/25/2016] [Indexed: 12/21/2022] Open
Abstract
Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications.
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Liu H, Zeng F, Zhang M, Huang F, Wang J, Guo J, Liu C, Wang H. Emerging landscape of cell penetrating peptide in reprogramming and gene editing. J Control Release 2016; 226:124-137. [PMID: 26849918 DOI: 10.1016/j.jconrel.2016.02.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 01/31/2016] [Accepted: 02/01/2016] [Indexed: 12/11/2022]
Abstract
The plasma membrane remains a major barrier for intracellular drug delivery, to overcome this issue, a variety of approaches have been developed and used to deliver therapeutic cargos. Among these approaches, cell penetrating peptide (CPP) is promising and affords widely used vector for efficient intracellular delivery of cargos. Moreover, the latter findings including iPS reprogramming and direct transdifferentiation as well as gene editing have gradually become hot research topic; because their application in tissue engineering and disease modeling have great potential to advance innovation in precision medicine. Since the beginning, research on these approaches is mainly based on virus transduction system, while, under the consideration for obviating the risk of mutagenic insertion and enables more accurate controlling, CPP-based efficient virus-free delivery strategy has been used recently. In this review, we summarize the existing CPP-based delivery system, emerging landscape of CPP application in stem cell manipulation and reprogramming, along with CPP contributions to gene editing techniques.
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Affiliation(s)
- Huiting Liu
- Medical School, China Three Gorges University, Yichang 443002, China; Department of Nuclear Medicine, Chongqing Three Gorges Central Hospital, Wanzhou 404000, China
| | - Fanhui Zeng
- The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, China
| | - Ming Zhang
- Medical School, China Three Gorges University, Yichang 443002, China
| | - Fajun Huang
- School of Medical Science, Hubei University for Nationalities, Enshi 445000, China
| | - Jiajun Wang
- Medical School, China Three Gorges University, Yichang 443002, China; School of Medical Science, Hubei University for Nationalities, Enshi 445000, China.
| | - Jingjing Guo
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States
| | - Changbai Liu
- Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang 443002, China.
| | - Hu Wang
- Medical School, China Three Gorges University, Yichang 443002, China; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
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Qin Y, Qin J, Zhou C, Li J, Gao WQ. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer. Cell Cycle 2016; 14:1282-90. [PMID: 25692793 DOI: 10.1080/15384101.2015.1007732] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
Abstract
Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.
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Affiliation(s)
- Yiren Qin
- a State Key Laboratory of Oncogenes and Related Genes; Renji-MedX Stem Cell Research Center; Ren Ji Hospital; School of Medicine ; Shanghai Jiao Tong University ; Shanghai , P.R. China
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Lewandowski J, Kolanowski TJ, Kurpisz M. Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes. J Tissue Eng Regen Med 2016; 11:1658-1674. [PMID: 26777594 DOI: 10.1002/term.2117] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Revised: 09/16/2015] [Accepted: 11/18/2015] [Indexed: 01/04/2023]
Abstract
The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post-infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC-mediated cardiomyocytes (iPSC-CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless-type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co-cultures with visceral endoderm-like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well-suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm-like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non-genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Jarosław Lewandowski
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Tomasz J Kolanowski
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Maciej Kurpisz
- Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
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Cellular Engineering and Disease Modeling with Gene-Editing Nucleases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_12] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells. Nat Commun 2015; 6:8036. [PMID: 26292731 PMCID: PMC4560784 DOI: 10.1038/ncomms9036] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2015] [Accepted: 07/06/2015] [Indexed: 02/07/2023] Open
Abstract
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. The expression of reprogramming factors can induce replication stress in induced pluripotent stem cells. In this study, to reduce such genomic instability, Ruiz et al. increase CHK1 kinase levels and nucleoside supplementation during reprogramming.
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38
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Recellularization of organs: what is the future for solid organ transplantation? Curr Opin Organ Transplant 2015; 19:603-9. [PMID: 25304814 DOI: 10.1097/mot.0000000000000131] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
PURPOSE OF REVIEW Allogeneic organ transplantation is burdened by donor shortage, graft rejection and adverse effects of lifelong immune suppression. Engineering bioartificial organs from acellular organ scaffolds and patient-derived cells are a new approach to potentially overcome these limitations. RECENT FINDINGS Decellularized organs yield a scaffold of extracellular matrix on which cells can adhere, integrate and ultimately form functional tissue. Various cell sources are currently used to repopulate acellular scaffolds, however, all have limitations. Patient-derived pluripotent stem cells hold great promise for tissue and organ engineering, when robust and mature cells can be directed in a reliable and safe manner. Finally, to produce mature organotypic tissue from a nonfunctional seeded scaffold, cellular scaffolds are cultured under biomimetic conditions in vitro. Alternatively, organs may be implanted at an immature stage to harness the recipient's body's regenerative capacity. In proof of principle experiments to date, bioengineered small animal organs have shown rudimentary function and maintained patency for limited time when transplanted in vivo. SUMMARY Recent advances in bioengineering organs raise the hope that we can overcome organ donor shortage and eliminate the need for livelong immunosuppression. However, significant challenges remain in generating mature large-scale donor-like bioartificial organs.
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Nityanandam A, Baldwin KK. Advances in reprogramming-based study of neurologic disorders. Stem Cells Dev 2015; 24:1265-83. [PMID: 25749371 DOI: 10.1089/scd.2015.0044] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The technology to convert adult human non-neural cells into neural lineages, through induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer, and direct lineage reprogramming or transdifferentiation has progressed tremendously in recent years. Reprogramming-based approaches aimed at manipulating cellular identity have enormous potential for disease modeling, high-throughput drug screening, cell therapy, and personalized medicine. Human iPSC (hiPSC)-based cellular disease models have provided proof of principle evidence of the validity of this system. However, several challenges remain before patient-specific neurons produced by reprogramming can provide reliable insights into disease mechanisms or be efficiently applied to drug discovery and transplantation therapy. This review will first discuss limitations of currently available reprogramming-based methods in faithfully and reproducibly recapitulating disease pathology. Specifically, we will address issues such as culture heterogeneity, interline and inter-individual variability, and limitations of two-dimensional differentiation paradigms. Second, we will assess recent progress and the future prospects of reprogramming-based neurologic disease modeling. This includes three-dimensional disease modeling, advances in reprogramming technology, prescreening of hiPSCs and creating isogenic disease models using gene editing.
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Affiliation(s)
- Anjana Nityanandam
- Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California
| | - Kristin K Baldwin
- Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California
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40
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Araki R, Sugiura M, Hoki Y, Sunayama M, Nakamura M, Kasama Y, Abe M. Induced pluripotent stem cell generation-associated point mutationsy. Inflamm Regen 2015. [DOI: 10.2492/inflammregen.35.226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Affiliation(s)
- Ryoko Araki
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Mayumi Sugiura
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Yuko Hoki
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Misato Sunayama
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Miki Nakamura
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Yasuji Kasama
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
| | - Masumi Abe
- Transcriptome Research Group, National Institute of Radiological Sciences, Chiba, Japan
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41
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Ben-David U. Genomic instability, driver genes and cell selection: Projections from cancer to stem cells. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2014; 1849:427-35. [PMID: 25132386 DOI: 10.1016/j.bbagrm.2014.08.005] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2014] [Revised: 07/03/2014] [Accepted: 08/02/2014] [Indexed: 12/13/2022]
Abstract
Cancer cells and stem cells share many traits, including a tendency towards genomic instability. Human cancers exhibit tumor-specific genomic aberrations, which often affect their malignancy and drug response. During their culture propagation, human pluripotent stem cells (hPSCs) also acquire characteristic genomic aberrations, which may have significant impact on their molecular and cellular phenotypes. These aberrations vary in size from single nucleotide alterations to copy number alterations to whole chromosome gains. A prominent challenge in both cancer and stem cell research is to identify "driver aberrations" that confer a selection advantage, and "driver genes" that underlie the recurrence of these aberrations. Following principles that are already well-established in cancer research, candidate driver genes have also been suggested in hPSCs. Experimental validation of the functional role of such candidates can uncover whether these are bona fide driver genes. The identification of driver genes may bring us closer to a mechanistic understanding of the genomic instability of stem cells. Guided by terminologies and methodologies commonly applied in cancer research, such understanding may have important ramifications for both stem cell and cancer biology. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
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Affiliation(s)
- Uri Ben-David
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
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42
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Ma H, Morey R, O'Neil RC, He Y, Daughtry B, Schultz MD, Hariharan M, Nery JR, Castanon R, Sabatini K, Thiagarajan RD, Tachibana M, Kang E, Tippner-Hedges R, Ahmed R, Gutierrez NM, Van Dyken C, Polat A, Sugawara A, Sparman M, Gokhale S, Amato P, Wolf DP, Ecker JR, Laurent LC, Mitalipov S. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 2014; 511:177-83. [PMID: 25008523 PMCID: PMC4898064 DOI: 10.1038/nature13551] [Citation(s) in RCA: 257] [Impact Index Per Article: 23.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 06/05/2014] [Indexed: 12/15/2022]
Abstract
Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.
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Affiliation(s)
- Hong Ma
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA [3]
| | - Robert Morey
- 1] Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037, USA [2]
| | - Ryan C O'Neil
- 1] Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA [2] Bioinformatics Program, University of California at San Diego, La Jolla, California 92093, USA
| | - Yupeng He
- 1] Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA [2] Bioinformatics Program, University of California at San Diego, La Jolla, California 92093, USA
| | - Brittany Daughtry
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Matthew D Schultz
- Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA
| | - Manoj Hariharan
- Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA
| | - Joseph R Nery
- Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA
| | - Rosa Castanon
- Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA
| | - Karen Sabatini
- Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037, USA
| | - Rathi D Thiagarajan
- Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037, USA
| | - Masahito Tachibana
- 1] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA [2] Department of Obstetrics and Gynecology, South Miyagi Medical Center, Shibata-gun, Miyagi 989-1253, Japan (M.T.); Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden (A.P.)
| | - Eunju Kang
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Rebecca Tippner-Hedges
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Riffat Ahmed
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Nuria Marti Gutierrez
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Crystal Van Dyken
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Alim Polat
- 1] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA [2] Department of Obstetrics and Gynecology, South Miyagi Medical Center, Shibata-gun, Miyagi 989-1253, Japan (M.T.); Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden (A.P.)
| | - Atsushi Sugawara
- Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Michelle Sparman
- Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Sumita Gokhale
- University Pathologists LLC, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island 02118, USA
| | - Paula Amato
- Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, USA
| | - Don P Wolf
- Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA
| | - Joseph R Ecker
- 1] Genomic Analysis Laboratory, the Salk Institute for Biological Studies, La Jolla, California 92037, USA [2] Howard Hughes Medical Institute, the Salk Institute for Biological Studies, La Jolla, California 92037, USA
| | - Louise C Laurent
- Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037, USA
| | - Shoukhrat Mitalipov
- 1] Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest Bond Avenue, Portland, Oregon 97239, USA [2] Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA [3] Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, USA
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Whole-genome sequencing identifies genetic variances in culture-expanded human mesenchymal stem cells. Stem Cell Reports 2014; 3:227-33. [PMID: 25254336 PMCID: PMC4176531 DOI: 10.1016/j.stemcr.2014.05.019] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Revised: 05/25/2014] [Accepted: 05/26/2014] [Indexed: 12/30/2022] Open
Abstract
Culture-expanded human mesenchymal stem cells (MSCs) are increasingly used in clinics, yet full characterization of the genomic compositions of these cells is lacking. We present a whole-genome investigation on the genetic dynamics of cultured MSCs under ex vivo establishment (passage 1 [p1]) and serial expansion (p8 and p13). We detected no significant changes in copy-number alterations (CNAs) and low levels of single-nucleotide changes (SNCs) until p8. Strikingly, a significant number (677) of SNCs were found in p13 MSCs. Using a sensitive Droplet Digital PCR assay, we tested the nonsynonymous SNCs detected by whole-genome sequencing and found that they were preexisting low-frequency mutations in uncultured mononuclear cells (∼0.01%) and early-passage MSCs (0.1%-1% at p1 and p8) but reached 17%-36% in p13. Our data demonstrate that human MSCs maintain a stable genomic composition in the early stages of ex vivo culture but are subject to clonal growth upon extended expansion.
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44
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Weissbein U, Benvenisty N, Ben-David U. Quality control: Genome maintenance in pluripotent stem cells. ACTA ACUST UNITED AC 2014; 204:153-63. [PMID: 24446481 PMCID: PMC3897183 DOI: 10.1083/jcb.201310135] [Citation(s) in RCA: 109] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Pluripotent stem cells (PSCs) must maintain their proper genomic content in order to preserve appropriate self-renewal and differentiation capacities. However, their prolonged in vitro propagation, as well as the environmental culture conditions, present serious challenges to genome maintenance. Recent work has been focused on potential means to alleviate the genomic insults experienced by PSCs, and to detect them as soon as they arise, in order to prevent the detrimental consequences of these genomic aberrations on PSC application in basic research and regenerative medicine.
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Affiliation(s)
- Uri Weissbein
- Stem Cell Unit, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel
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45
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Rio P, Baños R, Lombardo A, Quintana-Bustamante O, Alvarez L, Garate Z, Genovese P, Almarza E, Valeri A, Díez B, Navarro S, Torres Y, Trujillo JP, Murillas R, Segovia JC, Samper E, Surralles J, Gregory PD, Holmes MC, Naldini L, Bueren JA. Targeted gene therapy and cell reprogramming in Fanconi anemia. EMBO Mol Med 2014; 6:835-48. [PMID: 24859981 PMCID: PMC4203359 DOI: 10.15252/emmm.201303374] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.
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Affiliation(s)
- Paula Rio
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Rocio Baños
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Angelo Lombardo
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
| | - Oscar Quintana-Bustamante
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Lara Alvarez
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Zita Garate
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Pietro Genovese
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
| | - Elena Almarza
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Antonio Valeri
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Begoña Díez
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Susana Navarro
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | | | - Juan P Trujillo
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain
| | - Rodolfo Murillas
- Division of Epithelial Biomedicine, CIEMAT/CIBERER, Madrid, Spain
| | - Jose C Segovia
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | | | | | | | | | - Luigi Naldini
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy
| | - Juan A Bueren
- Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
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Li Z, Lu H, Yang W, Yong J, Zhang ZN, Zhang K, Deng H, Xu Y. Mouse SCNT ESCs have lower somatic mutation load than syngeneic iPSCs. Stem Cell Reports 2014; 2:399-405. [PMID: 24749065 PMCID: PMC3986627 DOI: 10.1016/j.stemcr.2014.02.005] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2013] [Revised: 02/14/2014] [Accepted: 02/17/2014] [Indexed: 12/21/2022] Open
Abstract
Ectopic expression of reprogramming factors has been widely adopted to reprogram somatic nucleus into a pluripotent state (induced pluripotent stem cells [iPSCs]). However, genetic aberrations such as somatic gene mutation in the resulting iPSCs have raised concerns regarding their clinical utility. To test whether the increased somatic mutations are primarily the by-products of current reprogramming methods, we reprogrammed embryonic fibroblasts of inbred C57BL/6 mice into either iPSCs (8 lines, 4 previously published) or embryonic stem cells (ESCs) with somatic cell nuclear transfer (SCNT ESCs; 11 lines). Exome sequencing of these lines indicates a significantly lower mutation load in SCNT ESCs than iPSCs of syngeneic background. In addition, one SCNT-ESC line has no detectable exome mutation, and two pairs of SCNT-ESC lines only have shared preexisting mutations. In contrast, every iPSC line carries unique mutations. Our study highlights the need for improving reprogramming methods in more physiologically relevant conditions.
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Affiliation(s)
- Zhe Li
- Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA ; Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA
| | - Hongxia Lu
- College of Life Sciences, Peking University, Beijing 100871, China
| | - Weifeng Yang
- College of Life Sciences, Peking University, Beijing 100871, China
| | - Jun Yong
- College of Life Sciences, Peking University, Beijing 100871, China
| | - Zhen-Ning Zhang
- Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA
| | - Kun Zhang
- Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA
| | - Hongkui Deng
- College of Life Sciences, Peking University, Beijing 100871, China
| | - Yang Xu
- Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA
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47
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Liang G, Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell 2014; 13:149-59. [PMID: 23910082 DOI: 10.1016/j.stem.2013.07.001] [Citation(s) in RCA: 281] [Impact Index Per Article: 25.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine. However, recent studies on the genetic and epigenetic variations in iPSCs have raised concerns that these variations may compromise the utility of iPSCs. In this Perspective, we review the current understanding of genetic and epigenetic variations in iPSCs, trace their causes, discuss the implications of these variations for iPSC applications, and propose approaches to cope with these variations.
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Affiliation(s)
- Gaoyang Liang
- Howard Hughes Medical Institute, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
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48
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Watanabe A, Amano N, Tokunaga Y, Poolsap U, Yamanaka S. Evaluation of safety of induced pluripotent stem cells by genome integrity. Inflamm Regen 2014. [DOI: 10.2492/inflammregen.34.087] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
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49
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Scheiner ZS, Talib S, Feigal EG. The potential for immunogenicity of autologous induced pluripotent stem cell-derived therapies. J Biol Chem 2013; 289:4571-7. [PMID: 24362036 DOI: 10.1074/jbc.r113.509588] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Induced pluripotent stem cell (iPSC) technology offers the promise of immune-matched cell therapies for a wide range of diseases and injuries. It is generally assumed that cells derived from autologous iPSCs will be immune-privileged. However, there are reasons to question this assumption, including recent studies that have tested iPSC immunogenicity in various ways with conflicting results. Understanding the risk of an immune response and developing strategies to minimize it will be important steps before clinical testing. Here, we review the evidence for autologous iPSC immunogenicity, its potential causes, and approaches for assessment and mitigation.
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Affiliation(s)
- Zachary S Scheiner
- From the California Institute for Regenerative Medicine, San Francisco, California 94107
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50
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Soyombo AA, Wu Y, Kolski L, Rios JJ, Rakheja D, Chen A, Kehler J, Hampel H, Coughran A, Ross TS. Analysis of induced pluripotent stem cells from a BRCA1 mutant family. Stem Cell Reports 2013; 1:336-49. [PMID: 24319668 PMCID: PMC3849250 DOI: 10.1016/j.stemcr.2013.08.004] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2013] [Revised: 08/14/2013] [Accepted: 08/15/2013] [Indexed: 02/09/2023] Open
Abstract
Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.
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Affiliation(s)
- Abigail A Soyombo
- Department of Internal Medicine, High Risk Cancer Genetics Program, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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