|
1
|
Gürhan G, Sevinç K, Aztekin C, Gayretli M, Yılmaz A, Yıldız AB, Ervatan EN, Morova T, Datlı E, Coleman OD, Kawamura A, Lack NA, Syed H, Önder T. A chromatin-focused CRISPR screen identifies USP22 as a barrier to somatic cell reprogramming. Commun Biol 2025; 8:454. [PMID: 40102626 PMCID: PMC11920211 DOI: 10.1038/s42003-025-07899-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 03/06/2025] [Indexed: 03/20/2025] Open
Abstract
Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen, we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly, this effect is likely to be independent of USP22's deubiquitinase activity or its association with the SAGA complex, as shown through module-specific knockouts, and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically, USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes, including DNMT3L, LIN28A, SOX2, and GDF3. Additionally, USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes, highlighting its potential as a target to improve reprogramming efficiency.
Collapse
Affiliation(s)
- Gülben Gürhan
- School of Medicine, Koç University, Istanbul, Turkey
| | - Kenan Sevinç
- School of Medicine, Koç University, Istanbul, Turkey
| | - Can Aztekin
- School of Medicine, Koç University, Istanbul, Turkey
| | - Mert Gayretli
- School of Medicine, Koç University, Istanbul, Turkey
| | | | | | | | - Tunç Morova
- School of Medicine, Koç University, Istanbul, Turkey
- Vancouver Prostate Centre, Department of Urologic Science, University of British Columbia, Vancouver, BC, Canada
| | - Elif Datlı
- School of Medicine, Koç University, Istanbul, Turkey
| | - Oliver D Coleman
- Chemistry - School of Natural and Environmental Sciences, Newcastle University, Newcastle, United Kingdom
| | - Akane Kawamura
- Chemistry - School of Natural and Environmental Sciences, Newcastle University, Newcastle, United Kingdom
| | - Nathan A Lack
- School of Medicine, Koç University, Istanbul, Turkey
- Vancouver Prostate Centre, Department of Urologic Science, University of British Columbia, Vancouver, BC, Canada
| | - Hamzah Syed
- School of Medicine, Koç University, Istanbul, Turkey
- Biostatistics, Bioinformatics and Data Management Core, KUTTAM, Koç University, Istanbul, Turkey
| | - Tamer Önder
- School of Medicine, Koç University, Istanbul, Turkey.
| |
Collapse
|
|
2
|
Yang J, Kinyamu HK, Ward JM, Scappini E, Muse G, Archer TK. Unlocking cellular plasticity: enhancing human iPSC reprogramming through bromodomain inhibition and extracellular matrix gene expression regulation. Stem Cells 2024; 42:706-719. [PMID: 38825983 PMCID: PMC11291304 DOI: 10.1093/stmcls/sxae039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Accepted: 05/15/2024] [Indexed: 06/04/2024]
Abstract
The transformation from a fibroblast mesenchymal cell state to an epithelial-like state is critical for induced pluripotent stem cell (iPSC) reprogramming. In this report, we describe studies with PFI-3, a small-molecule inhibitor that specifically targets the bromodomains of SMARCA2/4 and PBRM1 subunits of SWI/SNF complex, as an enhancer of iPSC reprogramming efficiency. Our findings reveal that PFI-3 induces cellular plasticity in multiple human dermal fibroblasts, leading to a mesenchymal-epithelial transition during iPSC formation. This transition is characterized by the upregulation of E-cadherin expression, a key protein involved in epithelial cell adhesion. Additionally, we identified COL11A1 as a reprogramming barrier and demonstrated COL11A1 knockdown increased reprogramming efficiency. Notably, we found that PFI-3 significantly reduced the expression of numerous extracellular matrix (ECM) genes, particularly those involved in collagen assembly. Our research provides key insights into the early stages of iPSC reprogramming, highlighting the crucial role of ECM changes and cellular plasticity in this process.
Collapse
Affiliation(s)
- Jun Yang
- Chromatin and Gene Expression Section, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| | - H Karimi Kinyamu
- Chromatin and Gene Expression Section, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| | - James M Ward
- Integrative Bioinformatics, Biostatistics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| | - Erica Scappini
- The Fluorescence Microscopy and Imaging Center, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| | - Ginger Muse
- Chromatin and Gene Expression Section, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| | - Trevor K Archer
- Chromatin and Gene Expression Section, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
| |
Collapse
|
|
3
|
Chowdhury MM, Zimmerman S, Leeson H, Nefzger CM, Mar JC, Laslett A, Polo JM, Wolvetang E, Cooper-White JJ. Superior Induced Pluripotent Stem Cell Generation through Phactr3-Driven Mechanomodulation of Both Early and Late Phases of Cell Reprogramming. Biomater Res 2024; 28:0025. [PMID: 38774128 PMCID: PMC11106629 DOI: 10.34133/bmr.0025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Accepted: 03/25/2024] [Indexed: 05/24/2024] Open
Abstract
Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes.
Collapse
Affiliation(s)
- Mohammad Mahfuz Chowdhury
- Australian Institute of Bioengineering and Nanotechnology (AIBN),
The University of Queensland, St. Lucia, QLD 4072, Australia
| | | | - Hannah Leeson
- Australian Institute of Bioengineering and Nanotechnology (AIBN),
The University of Queensland, St. Lucia, QLD 4072, Australia
| | | | - Jessica Cara Mar
- Australian Institute of Bioengineering and Nanotechnology (AIBN),
The University of Queensland, St. Lucia, QLD 4072, Australia
- Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Andrew Laslett
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia
| | - Jose Maria Polo
- Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute and the Australian Regenerative Medicine Institute,
Monash University, Clayton, VIC 3800, Australia
- Adelaide Centre for Epigenetics and the South Australian Immunogenomics Cancer Institute, Faculty of Health and Medical Sciences,
The University of Adelaide, Adelaide, SA 5005, Australia
| | - Ernst Wolvetang
- Australian Institute of Bioengineering and Nanotechnology (AIBN),
The University of Queensland, St. Lucia, QLD 4072, Australia
| | - Justin John Cooper-White
- Australian Institute of Bioengineering and Nanotechnology (AIBN),
The University of Queensland, St. Lucia, QLD 4072, Australia
- School of Chemical Engineering, Andrew N. Liveris Building,
The University of Queensland, St. Lucia, QLD 4072, Australia
| |
Collapse
|
|
4
|
Swaidan NT, Soliman NH, Aboughalia AT, Darwish T, Almeshal RO, Al-Khulaifi AA, Taha RZ, Alanany R, Hussein AY, Salloum-Asfar S, Abdulla SA, Abdallah AM, Emara MM. CCN3, POSTN, and PTHLH as potential key regulators of genomic integrity and cellular survival in iPSCs. Front Mol Biosci 2024; 11:1342011. [PMID: 38375508 PMCID: PMC10875024 DOI: 10.3389/fmolb.2024.1342011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Accepted: 01/11/2024] [Indexed: 02/21/2024] Open
Abstract
Reprogramming human somatic cells into a pluripotent state, achieved through the activation of well-defined transcriptional factors known as OSKM factors, offers significant potential for regenerative medicine. While OSKM factors are a robust reprogramming method, efficiency remains a challenge, with only a fraction of cells undergoing successful reprogramming. To address this, we explored genes related to genomic integrity and cellular survival, focusing on iPSCs (A53T-PD1) that displayed enhanced colony stability. Our investigation had revealed three candidate genes CCN3, POSTN, and PTHLH that exhibited differential expression levels and potential roles in iPSC stability. Subsequent analyses identified various protein interactions for these candidate genes. POSTN, significantly upregulated in A53T-PD1 iPSC line, showed interactions with extracellular matrix components and potential involvement in Wnt signaling. CCN3, also highly upregulated, demonstrated interactions with TP53, CDKN1A, and factors related to apoptosis and proliferation. PTHLH, while upregulated, exhibited interactions with CDK2 and genes involved in cell cycle regulation. RT-qPCR validation confirmed elevated CCN3 and PTHLH expression in A53T-PD1 iPSCs, aligning with RNA-seq findings. These genes' roles in preserving pluripotency and cellular stability require further exploration. In conclusion, we identified CCN3, POSTN, and PTHLH as potential contributors to genomic integrity and pluripotency maintenance in iPSCs. Their roles in DNA repair, apoptosis evasion, and signaling pathways could offer valuable insights for enhancing reprogramming efficiency and sustaining pluripotency. Further investigations are essential to unravel the mechanisms underlying their actions.
Collapse
Affiliation(s)
- Nuha T. Swaidan
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Nada H. Soliman
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Ahmed T. Aboughalia
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Toqa Darwish
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Ruba O. Almeshal
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Azhar A. Al-Khulaifi
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Rowaida Z. Taha
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Rania Alanany
- College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar
| | | | - Salam Salloum-Asfar
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Sara A. Abdulla
- Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
| | - Abdallah M. Abdallah
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| | - Mohamed M. Emara
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, Doha, Qatar
| |
Collapse
|
|
5
|
Bekas N, Samiotaki M, Papathanasiou M, Mokos P, Pseftogas A, Xanthopoulos K, Thanos D, Mosialos G, Dafou D. Inactivation of Tumor Suppressor CYLD Inhibits Fibroblast Reprogramming to Pluripotency. Cancers (Basel) 2023; 15:4997. [PMID: 37894364 PMCID: PMC10605754 DOI: 10.3390/cancers15204997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2023] [Accepted: 10/12/2023] [Indexed: 10/29/2023] Open
Abstract
CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLDΔ9/Δ9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLDΔ9/Δ9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.
Collapse
Affiliation(s)
- Nikolaos Bekas
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Martina Samiotaki
- Biomedical Sciences Research Center “Alexander Fleming”, 16672 Vari, Greece;
| | - Maria Papathanasiou
- Biomedical Research Foundation Academy of Athens, 11527 Athens, Greece; (M.P.); (D.T.)
| | - Panagiotis Mokos
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Athanasios Pseftogas
- Division of Experimental Oncology, IRCCS San Raffaele Hospital, Vita-Salute San Raffaele University, 20132 Milan, Italy;
| | - Konstantinos Xanthopoulos
- Laboratory of Pharmacology, Department of Pharmacy, School of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Dimitris Thanos
- Biomedical Research Foundation Academy of Athens, 11527 Athens, Greece; (M.P.); (D.T.)
| | - George Mosialos
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| | - Dimitra Dafou
- School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (N.B.); (P.M.); (G.M.)
| |
Collapse
|
|
6
|
Yang J, Karimi Kinyamu H, Ward JM, Scappini E, Archer TK. Unlocking cellular plasticity: Enhancing human iPSC reprogramming through bromodomain inhibition and extracellular matrix gene expression regulation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.13.562265. [PMID: 37873209 PMCID: PMC10592827 DOI: 10.1101/2023.10.13.562265] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
The transformation of fibroblasts into epithelial cells is critical for iPSC reprogramming. In this report, we describe studies with PFI-3, a small molecule inhibitor that specifically targets the bromodomains of SMARCA2/4 and PBRM1 subunit of SWI/SNF complex, as an enhancer of iPSC reprogramming efficiency. Our findings revealed that PFI-3 induces cellular plasticity in multiple human dermal fibroblasts, leading to a mesenchymal-epithelial transition (MET) during iPSC formation. This transition was characterized by the upregulation of E-cadherin expression, a key protein involved in epithelial cell adhesion. Additionally, we identified COL11A1 as a reprogramming barrier and demonstrated COL11A1 knockdown increased reprogramming efficiency. Notably, we found that PFI-3 significantly reduced the expression of numerous extracellular matrix (ECM) genes, particularly those involved in collagen assembly. Our research provides key insights into the early stages of iPSC reprogramming, highlighting the crucial role of ECM changes and cellular plasticity in this process.
Collapse
|
|
7
|
Du J, Yuan X, Deng H, Huang R, Liu B, Xiong T, Long X, Zhang L, Li Y, She Q. Single-cell and spatial heterogeneity landscapes of mature epicardial cells. J Pharm Anal 2023; 13:894-907. [PMID: 37719196 PMCID: PMC10499659 DOI: 10.1016/j.jpha.2023.07.011] [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: 11/28/2022] [Revised: 07/13/2023] [Accepted: 07/18/2023] [Indexed: 09/19/2023] Open
Abstract
Tbx18, Wt1, and Tcf21 have been identified as epicardial markers during the early embryonic stage. However, the gene markers of mature epicardial cells remain unclear. Single-cell transcriptomic analysis was performed with the Seurat, Monocle, and CellphoneDB packages in R software with standard procedures. Spatial transcriptomics was performed on chilled Visium Tissue Optimization Slides (10x Genomics) and Visium Spatial Gene Expression Slides (10x Genomics). Spatial transcriptomics analysis was performed with Space Ranger software and R software. Immunofluorescence, whole-mount RNA in situ hybridization and X-gal staining were performed to validate the analysis results. Spatial transcriptomics analysis revealed distinct transcriptional profiles and functions between epicardial tissue and non-epicardial tissue. Several gene markers specific to postnatal epicardial tissue were identified, including Msln, C3, Efemp1, and Upk3b. Single-cell transcriptomic analysis revealed that cardiac cells from wildtype mouse hearts (from embryonic day 9.5 to postnatal day 9) could be categorized into six major cell types, which included epicardial cells. Throughout epicardial development, Wt1, Tbx18, and Upk3b were consistently expressed, whereas genes including Msln, C3, and Efemp1 exhibited increased expression during the mature stages of development. Pseudotime analysis further revealed two epicardial cell fates during maturation. Moreover, Upk3b, Msln, Efemp1, and C3 positive epicardial cells were enriched in extracellular matrix signaling. Our results suggested Upk3b, Efemp1, Msln, C3, and other genes were mature epicardium markers. Extracellular matrix signaling was found to play a critical role in the mature epicardium, thus suggesting potential therapeutic targets for heart regeneration in future clinical practice.
Collapse
Affiliation(s)
- Jianlin Du
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Xin Yuan
- Department of Nephrology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Haijun Deng
- Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Institute for Viral Hepatitis, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Rongzhong Huang
- Precision Medicine Center, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Bin Liu
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Tianhua Xiong
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Xianglin Long
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Ling Zhang
- Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Yingrui Li
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| | - Qiang She
- Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China
| |
Collapse
|
|
8
|
Yang BA, da Rocha AM, Newton I, Shcherbina A, Wong SW, Fraczek PM, Larouche JA, Hiraki HL, Baker BM, Shin JW, Takayama S, Thouless MD, Aguilar CA. Manipulation of the nucleoscaffold potentiates cellular reprogramming kinetics. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.12.532246. [PMID: 36993714 PMCID: PMC10055010 DOI: 10.1101/2023.03.12.532246] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 06/19/2023]
Abstract
Somatic cell fate is an outcome set by the activities of specific transcription factors and the chromatin landscape and is maintained by gene silencing of alternate cell fates through physical interactions with the nuclear scaffold. Here, we evaluate the role of the nuclear scaffold as a guardian of cell fate in human fibroblasts by comparing the effects of transient loss (knockdown) and mutation (progeria) of functional Lamin A/C, a core component of the nuclear scaffold. We observed that Lamin A/C deficiency or mutation disrupts nuclear morphology, heterochromatin levels, and increases access to DNA in lamina-associated domains. Changes in Lamin A/C were also found to impact the mechanical properties of the nucleus when measured by a microfluidic cellular squeezing device. We also show that transient loss of Lamin A/C accelerates the kinetics of cellular reprogramming to pluripotency through opening of previously silenced heterochromatin domains while genetic mutation of Lamin A/C into progerin induces a senescent phenotype that inhibits the induction of reprogramming genes. Our results highlight the physical role of the nuclear scaffold in safeguarding cellular fate.
Collapse
Affiliation(s)
- Benjamin A. Yang
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA
| | | | - Isabel Newton
- Dept. of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Anna Shcherbina
- Dept. of Biomedical Informatics, Stanford University, Palo Alto, CA 94305, USA
| | - Sing-Wan Wong
- Dept. of Pharmacology and Biomedical Engineering, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Paula M. Fraczek
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA
| | - Jacqueline A. Larouche
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA
| | - Harrison L. Hiraki
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Brendon M. Baker
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Jae-Won Shin
- Dept. of Pharmacology and Biomedical Engineering, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Shuichi Takayama
- Wallace Coulter Dept. of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332
| | - M. D. Thouless
- Dept. of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Dept. of Materials Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Carlos A. Aguilar
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA
- Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA
| |
Collapse
|
|
9
|
Sun W, Zhang S, Zhou T, Shan Y, Gao F, Zhang Y, Zhang D, Xiong Y, Mai Y, Fan K, Davidson AJ, Pan G, Zhang X. Human Urinal Cell Reprogramming: Synthetic 3D Peptide Hydrogels Enhance Induced Pluripotent Stem Cell Population Homogeneity. ACS Biomater Sci Eng 2020; 6:6263-6275. [PMID: 33449655 DOI: 10.1021/acsbiomaterials.0c00667] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which have promising potential applications in regenerative medicine. However, the challenges of successful applications of human iPSCs for medical purposes are the low generation efficiency, heterogeneous colonies, and exposure to the animal-derived product Matrigel. We aimed to investigate whether human urinal cells could be efficiently reprogrammed into iPSCs in three-dimensional Puramatrix (3D-PM) compared to two-dimensional Matrigel (2D-MG) and to understand how this 3D hydrogel environment affects the reprogramming process. Human urinal cells were successfully reprogrammed into iPSCs in the defined synthetic animal-free 3D-PM. Interestingly, although the colony efficiency in 3D-PM was similar to that in 2D-MG (∼0.05%), the reprogrammed colonies in 3D-PM contained an iPSC population with significantly higher homogeneity, as evidenced by the pluripotent-like morphology and expression of markers. This was further confirmed by transcriptome profile analysis in bulk cells and at the single cell level. Moreover, the homogeneity of the iPSC population in 3D-PM colonies was correlated with the downregulation of integrin β1 (ITGB1) and phosphorylated focal adhesion kinase (FAK). Collectively, 3D-PM provides an alternative approach for obtaining iPSCs with enhanced homogeneity. This work also unveiled the regulation of human somatic cell reprogramming via the extracellular microenvironment.
Collapse
Affiliation(s)
- Wei Sun
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Sheng Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Tiancheng Zhou
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yongli Shan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Fenglin Gao
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610051, China
| | - Ying Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Di Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yucui Xiong
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yuanbang Mai
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Ke Fan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Alan J Davidson
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Guangjin Pan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Xiao Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Guangzhou Medical University, Guangzhou 511436, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| |
Collapse
|
|
10
|
Xing QR, El Farran CA, Gautam P, Chuah YS, Warrier T, Toh CXD, Kang NY, Sugii S, Chang YT, Xu J, Collins JJ, Daley GQ, Li H, Zhang LF, Loh YH. Diversification of reprogramming trajectories revealed by parallel single-cell transcriptome and chromatin accessibility sequencing. SCIENCE ADVANCES 2020; 6:eaba1190. [PMID: 32917699 PMCID: PMC7486102 DOI: 10.1126/sciadv.aba1190] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/06/2019] [Accepted: 07/30/2020] [Indexed: 05/16/2023]
Abstract
Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery.
Collapse
Affiliation(s)
- Q R Xing
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore
| | - Chadi A El Farran
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
| | - Pradeep Gautam
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
| | - Yu Song Chuah
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
| | - Tushar Warrier
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
| | - Cheng-Xu Delon Toh
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
| | - Nam-Young Kang
- Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, A*STAR, Singapore 138667, Singapore
- Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Shigeki Sugii
- Institute of Bioengineering and Nanotechnology, A*STAR, Singapore 138669, Singapore
- Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, Singapore 169857, Singapore
| | - Young-Tae Chang
- Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, A*STAR, Singapore 138667, Singapore
- Department of Chemistry, National University of Singapore, Singapore 117543, Singapore
- Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang 37673, Republic of Korea
- Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Jian Xu
- Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
- Department of Plant Systems Physiology, Institute for Water and Wetland Research, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, Netherlands
| | - James J Collins
- Institute for Medical Engineering and Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
| | - George Q Daley
- Stem Cell Program, Division of Pediatric Hematology and Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115, USA
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Cambridge, MA 02138, USA
- Manton Center for Orphan Disease Research, Boston, MA 02115, USA
| | - Hu Li
- Center for Individualized Medicine, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
| | - Li-Feng Zhang
- School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
| | - Yuin-Han Loh
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.
- Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 119077, Singapore
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore
| |
Collapse
|
|
11
|
Al-Moujahed A, Tian B, Efstathiou NE, Konstantinou EK, Hoang M, Lin H, Miller JW, Vavvas DG. Receptor interacting protein kinase 3 (RIP3) regulates iPSCs generation through modulating cell cycle progression genes. Stem Cell Res 2019; 35:101387. [PMID: 30703581 PMCID: PMC7375132 DOI: 10.1016/j.scr.2019.101387] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2018] [Revised: 12/24/2018] [Accepted: 01/18/2019] [Indexed: 01/02/2023] Open
Abstract
The molecular mechanisms involved in induced pluripotent stem cells (iPSCs) generation are poorly understood. The cell death machinery of apoptosis-inducing caspases have been shown to facilitate the process of iPSCs reprogramming. However, the effect of other cell death processes, such as programmed necrosis (necroptosis), on iPSCs induction has not been studied. In this study, we investigated the role of receptor-interacting protein kinase 3 (RIP3), an essential regulator of necroptosis, in reprogramming mouse embryonic fibroblast cells (MEFs) into iPSCs. RIP3 was found to be upregulated in iPSCs compared to MEFs. Deletion of RIP3 dramatically suppressed the reprogramming of iPSCs (~82%). RNA-seq analysis and qRT-PCR showed that RIP3 KO MEFs expressed lower levels of genes that control cell cycle progression and cell division and higher levels of extracellular matrix-regulating genes. The growth rate of RIP3 KO MEFs was significantly slower than WT MEFs. These findings can partially explain the inhibitory effects of RIP3 deletion on iPSCs generation and show for the first time that the necroptosis kinase RIP3 plays an important role in iPSC reprogramming. In contrast to RIP3, the kinase and scaffolding functions of RIPK1 appeared to have distinct effects on reprogramming.
Collapse
Affiliation(s)
- Ahmad Al-Moujahed
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States; Department of Pathology, Boston University School of Medicine, Boston, MA 02118, United States
| | - Bo Tian
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States; Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, MA 01605, United States
| | - Nikolaos E Efstathiou
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States
| | - Eleni K Konstantinou
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States
| | - Mien Hoang
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States
| | - Haijiang Lin
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States; Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, MA 01605, United States
| | - Joan W Miller
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States
| | - Demetrios G Vavvas
- Retina Service, Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, United States.
| |
Collapse
|
|
12
|
Xu P, Wang TT, Liu XZ, Wang NY, Sun LH, Zhang ZQ, Chen HZ, Lv X, Huang Y, Liu DP. Sirt6 regulates efficiency of mouse somatic reprogramming and maintenance of pluripotency. Stem Cell Res Ther 2019; 10:9. [PMID: 30630525 PMCID: PMC6329104 DOI: 10.1186/s13287-018-1109-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2018] [Revised: 12/04/2018] [Accepted: 12/13/2018] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Mouse somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. It has been reported that Sirtuin 6 (Sirt6), a member of the sirtuin family of NAD+-dependent protein deacetylases, is involved in embryonic stem cell differentiation. However, whether and how Sirt6 influences epigenetic reprogramming remains unknown. METHODS Mouse embryonic fibroblasts isolated from transgenic Oct4-GFP reporter mice with or without Sirt6 were used for reprogramming by Yamanaka factors. Alkaline phosphatase-positive and OCT4-GFP-positive colony were counted to calculate reprogramming efficiency. OP9 feeder cell co-culture system was used to measure the hematopoietic differentiation from mouse ES and iPS cells. RNA sequencing was measured to identify the differential expressed genes due to loss of Sirt6 in somatic and pluripotent cells. RESULTS In this study, we provide evidence that Sirt6 is involved in mouse somatic reprogramming. We found that loss of function of Sirt6 could significantly decrease reprogramming efficiency. Furthermore, we showed that Sirt6-null iPS-like cell line has intrinsically a differentiation defect even though the establishment of normal self-renewal. Particularly, by performing transcriptome analysis, we observed that several pluripotent transcriptional factors increase in knockout cell line, which explains the underlying loss of pluripotency in Sirt6-null iPS-like cell line. CONCLUSIONS Taken together, we have identified a new regulatory role of Sirt6 in reprogramming and maintenance of pluripotency.
Collapse
Affiliation(s)
- Peng Xu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Ting-ting Wang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Central Research Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730 China
| | - Xiu-zhen Liu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Nan-Yu Wang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Li-hong Sun
- Center for Experimental Animal Research, Institute of Basic Medical Sciences Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005 China
| | - Zhu-qin Zhang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Hou-zao Chen
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Xiang Lv
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| | - Yue Huang
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005 China
| | - De-Pei Liu
- State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
- Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005 China
| |
Collapse
|
|
13
|
FACT Sets a Barrier for Cell Fate Reprogramming in Caenorhabditis elegans and Human Cells. Dev Cell 2018; 46:611-626.e12. [PMID: 30078731 PMCID: PMC6137076 DOI: 10.1016/j.devcel.2018.07.006] [Citation(s) in RCA: 64] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Revised: 05/08/2018] [Accepted: 07/03/2018] [Indexed: 01/04/2023]
Abstract
The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection.
Collapse
|
|
14
|
Jiao J, Tian W, Qiu P, Norton EL, Wang MM, Chen YE, Yang B. Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy. J Thorac Cardiovasc Surg 2018; 156:515-522.e1. [PMID: 29653750 PMCID: PMC9809054 DOI: 10.1016/j.jtcvs.2018.02.087] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2017] [Revised: 01/26/2018] [Accepted: 02/02/2018] [Indexed: 02/07/2023]
Abstract
OBJECTIVE The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. METHODS The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1-/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1-/- and wild type cells. RESULTS The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1-/-NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1-/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1-/-CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1-/-ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. CONCLUSIONS NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.
Collapse
Affiliation(s)
- Jiao Jiao
- Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich
| | - Weihua Tian
- Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich
| | - Ping Qiu
- Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich
| | | | - Michael M. Wang
- Department of Neurology, Michigan Medicine, Ann Arbor, Mich;,VA Ann Arbor Healthcare System, Ann Arbor, Mich
| | - Y. Eugene Chen
- Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich
| | - Bo Yang
- Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich
| |
Collapse
|
|
15
|
Chen X, Li J, Huang Y, Liu P, Fan Y. Insoluble Microenvironment Facilitating the Generation and Maintenance of Pluripotency. TISSUE ENGINEERING PART B-REVIEWS 2018; 24:267-278. [PMID: 29327674 DOI: 10.1089/ten.teb.2017.0415] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Induced pluripotent stem cells (iPSCs) hold enormous potential as a tool to generate cells for tissue engineering and regenerative medicine. Since the initial report of iPSCs in 2006, many different methods have been developed to enhance the safety and efficiency of this technology. Recent studies indicate that the extracellular signals can promote the production of iPSCs, and even replace the Yamanaka factors. Noticeably, abundant evidences suggest that the insoluble microenvironment, including the culture substrate and neighboring cells, directly regulates the expression of core pluripotency genes and the epigenetic modification of the chromatins, hence, impacts the reprogramming dynamics. These studies provide new strategies for developing safer and more efficient method for iPSC generation. In this review, we examine the publications addressing the insoluble extracellular microenvironment that boosts iPSC generation and self-renewal. We also discuss cell adhesion-mediated molecular mechanisms, through which the insoluble extracellular cues interplay with reprogramming.
Collapse
Affiliation(s)
- Xiaofang Chen
- 1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University , Beijing, China
- 2 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University , Beijing, China
| | - Jiaqi Li
- 1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University , Beijing, China
| | - Yan Huang
- 1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University , Beijing, China
- 2 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University , Beijing, China
| | - Peng Liu
- 3 Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University , Beijing, China
| | - Yubo Fan
- 1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University , Beijing, China
- 2 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University , Beijing, China
- 4 National Research Center for Rehabilitation Technical Aids , Beijing, China
| |
Collapse
|
|
16
|
Fang HT, El Farran CA, Xing QR, Zhang LF, Li H, Lim B, Loh YH. Global H3.3 dynamic deposition defines its bimodal role in cell fate transition. Nat Commun 2018; 9:1537. [PMID: 29670118 PMCID: PMC5906632 DOI: 10.1038/s41467-018-03904-7] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2017] [Accepted: 03/21/2018] [Indexed: 01/19/2023] Open
Abstract
H3.3 is a histone variant, which is deposited on genebodies and regulatory elements, by Hira, marking active transcription. Moreover, H3.3 is deposited on heterochromatin by Atrx/Daxx complex. The exact role of H3.3 in cell fate transition remains elusive. Here, we investigate the dynamic changes in the deposition of the histone variant H3.3 during cellular reprogramming. H3.3 maintains the identities of the parental cells during reprogramming as its removal at early time-point enhances the efficiency of the process. We find that H3.3 plays a similar role in transdifferentiation to hematopoietic progenitors and neuronal differentiation from embryonic stem cells. Contrastingly, H3.3 deposition on genes associated with the newly reprogrammed lineage is essential as its depletion at the later phase abolishes the process. Mechanistically, H3.3 deposition by Hira, and its K4 and K36 modifications are central to the role of H3.3 in cell fate conversion. Finally, H3.3 safeguards fibroblast lineage by regulating Mapk cascade and collagen synthesis.
Collapse
Affiliation(s)
- Hai-Tong Fang
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Ageing, A*STAR Institute of Molecular and Cell Biology, Singapore, 138673, Singapore
| | - Chadi A El Farran
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Ageing, A*STAR Institute of Molecular and Cell Biology, Singapore, 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore
| | - Qiao Rui Xing
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Ageing, A*STAR Institute of Molecular and Cell Biology, Singapore, 138673, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore
| | - Li-Feng Zhang
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore
| | - Hu Li
- Center for Individualized Medicine, Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN, 55905, USA.
| | - Bing Lim
- Stem Cell and Regenerative Biology Group, Genome Institute of Singapore, Singapore, 138672, Singapore
| | - Yuin-Han Loh
- Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Ageing, A*STAR Institute of Molecular and Cell Biology, Singapore, 138673, Singapore.
- Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore.
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, 117456, Singapore.
| |
Collapse
|
|
17
|
Pizzute T, Li J, Zhang Y, Davis ME, Pei M. Fibroblast Growth Factor Ligand Dependent Proliferation and Chondrogenic Differentiation of Synovium-Derived Stem Cells and Concomitant Adaptation of Wnt/Mitogen-Activated Protein Kinase Signals. Tissue Eng Part A 2017; 22:1036-46. [PMID: 27411850 DOI: 10.1089/ten.tea.2016.0102] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cell expansion techniques commonly utilize exogenous factors to increase cell proliferation and create a larger cell population for use in cell-based therapies. One strategy for cartilage regenerative therapies is autologous stem cell expansion and fibroblast growth factor (FGF) supplementation during cell expansion, particularly FGF-2. However, it is unknown whether FGF-10, another FGF implicated in limb and skeletal development, can elicit the same rejuvenation responses in terms of proliferation and differentiation of human synovium-derived stem cells (SDSCs). In this study, we expanded SDSCs in either FGF-2 or FGF-10 for 7 days; a control group had no treatment. FGF-2 and FGF-10 supplementation was also exclusively tested during the differentiation phase. Expanded SDSCs were evaluated for their ability to successfully engage in chondrogenic and osteogenic differentiation. We found that FGF-2 supplementation during proliferation, but not differentiation, was able to increase glycosaminoglycan deposition, pellet size, and chondrogenic gene expression following chondrogenic induction, as well as increased calcium deposition, alkaline phosphatase activity, and expression of vital osteogenic differentiation genes following osteogenic induction. FGF-10 did not elicit a similar preconditioning effect. We also observed changes of both Wnt signals and mitogen-activated protein kinase expression during SDSC chondrogenesis, which occurred in a manner dependent upon the supplementation phase of FGF-2 administration. These results indicated that FGF-2, but not FGF-10, may be supplemented during stem cell expansion to prime cells for successful chondrogenesis and osteogenesis.
Collapse
Affiliation(s)
- Tyler Pizzute
- 1 Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University , Morgantown, West Virginia.,2 Exercise Physiology, West Virginia University , Morgantown, West Virginia
| | - Jingting Li
- 1 Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University , Morgantown, West Virginia.,2 Exercise Physiology, West Virginia University , Morgantown, West Virginia
| | - Ying Zhang
- 1 Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University , Morgantown, West Virginia.,3 Mechanical and Aerospace Engineering, West Virginia University , Morgantown, West Virginia
| | - Mary E Davis
- 4 Department of Physiology and Pharmacology, West Virginia University , Morgantown, West Virginia
| | - Ming Pei
- 1 Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University , Morgantown, West Virginia.,2 Exercise Physiology, West Virginia University , Morgantown, West Virginia.,3 Mechanical and Aerospace Engineering, West Virginia University , Morgantown, West Virginia
| |
Collapse
|
|
18
|
Moradi S, Sharifi-Zarchi A, Ahmadi A, Mollamohammadi S, Stubenvoll A, Günther S, Salekdeh GH, Asgari S, Braun T, Baharvand H. Small RNA Sequencing Reveals Dlk1-Dio3 Locus-Embedded MicroRNAs as Major Drivers of Ground-State Pluripotency. Stem Cell Reports 2017; 9:2081-2096. [PMID: 29129685 PMCID: PMC5785679 DOI: 10.1016/j.stemcr.2017.10.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2016] [Revised: 10/11/2017] [Accepted: 10/11/2017] [Indexed: 11/26/2022] Open
Abstract
Ground-state pluripotency is a cell state in which pluripotency is established and maintained through efficient repression of endogenous differentiation pathways. Self-renewal and pluripotency of embryonic stem cells (ESCs) are influenced by ESC-associated microRNAs (miRNAs). Here, we provide a comprehensive assessment of the “miRNome” of ESCs cultured under conditions favoring ground-state pluripotency. We found that ground-state ESCs express a distinct set of miRNAs compared with ESCs grown in serum. Interestingly, most “ground-state miRNAs” are encoded by an imprinted region on chromosome 12 within the Dlk1-Dio3 locus. Functional analysis revealed that ground-state miRNAs embedded in the Dlk1-Dio3 locus (miR-541-5p, miR-410-3p, and miR-381-3p) promoted pluripotency via inhibition of multi-lineage differentiation and stimulation of self-renewal. Overall, our results demonstrate that ground-state pluripotency is associated with a unique miRNA signature, which supports ground-state self-renewal by suppressing differentiation.
Ground-state pluripotency is associated with a unique miRNA signature Dlk1-Dio3 locus on 12qF1 encodes the majority of ground-state miRNAs Dlk1-Dio3 locus is hypomethylated in ground-state ESCs Ground-state miRNAs promote ESC self-renewal and inhibit differentiation
Collapse
Affiliation(s)
- Sharif Moradi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Ali Sharifi-Zarchi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran; Computer Engineering Department, Sharif University of Technology, Tehran, Iran
| | - Amirhossein Ahmadi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran
| | - Sepideh Mollamohammadi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran
| | - Alexander Stubenvoll
- Max-Planck Institute for Heart and Lung Research, Department of Cardiac Development and Remodelling, Ludwigstrasse 43, 61231 Bad Nauheim, Germany
| | - Stefan Günther
- Max-Planck Institute for Heart and Lung Research, Department of Cardiac Development and Remodelling, Ludwigstrasse 43, 61231 Bad Nauheim, Germany
| | - Ghasem Hosseini Salekdeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran
| | - Sassan Asgari
- Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD, Australia
| | - Thomas Braun
- Max-Planck Institute for Heart and Lung Research, Department of Cardiac Development and Remodelling, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Ressalat Highway, Tehran 1665659911, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
| |
Collapse
|
|
19
|
Common microRNA-mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states. Cell Death Dis 2017; 8:e3027. [PMID: 29048434 PMCID: PMC5596602 DOI: 10.1038/cddis.2017.426] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Revised: 07/17/2017] [Accepted: 07/25/2017] [Indexed: 12/11/2022]
Abstract
Previous evidences have proved that porcine-induced pluripotent stem cells (piPSCs) could be induced to distinctive metastable pluripotent states. This raises the issue of whether there is a common transcriptomic profile existing among the piPSC lines at distinctive state. In this study, we performed conjoint analysis of small RNA-seq and mRNA-seq for three piPSC lines which represent LIF dependence, FGF2 dependence and LFB2i dependence, respectively. Interestingly, we found there are 16 common microRNAs which potentially target 13 common mRNAs among the three piPSC lines. Dual-luciferase reporter assay validated that miR-370, one of the 16 common microRNAs, could directly target the 3′UTR of LIN28A. When the differentiation occurred, miR-370 could be activated in piPSCs and switched off the expression of LIN28A. Ectopic expression of miR-370 in piPSCs could reduce LIN28A expression, decrease the alkaline phosphatase activity, slow down the proliferation, and further cause the downregulation of downstream pluripotent genes (OCT4, SOX2, NANOG, SALL4 and ESRRB) and upregulation of differentiation relevant genes (SOX9, JARID2 and JMJD4). Moreover, these phenotypes caused by miR-370 could be rescued by overexpressing LIN28A. Collectively, our findings suggest that a set of common miRNA–mRNA interactions exist among the distinct piPSC lines, which orchestrate the self-renewal and differentiation of piPSCs independent of their metastable pluripotent states.
Collapse
|
|
20
|
Rawat N, Singh MK. Induced pluripotent stem cell: A headway in reprogramming with promising approach in regenerative biology. Vet World 2017; 10:640-649. [PMID: 28717316 PMCID: PMC5499081 DOI: 10.14202/vetworld.2017.640-649] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2017] [Accepted: 04/26/2017] [Indexed: 12/17/2022] Open
Abstract
Since the embryonic stem cells have knocked the doorsteps, they have proved themselves in the field of science, research, and medicines, but the hovered restrictions confine their application in human welfare. Alternate approaches used to reprogram the cells to the pluripotent state were not up to par, but the innovation of induced pluripotent stem cells (iPSCs) paved a new hope for the researchers. Soon after the discovery, iPSCs technology is undergoing renaissance day by day, i.e., from the use of genetic material to recombinant proteins and now only chemicals are employed to convert somatic cells to iPSCs. Thus, this technique is moving straightforward and productive at an astonishing pace. Here, we provide a brief introduction to iPSCs, the mechanism and methods for their generation, their prevailing and prospective applications and the future opportunities that can be expected from them.
Collapse
Affiliation(s)
- N Rawat
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR - National Dairy Research Institute, Karnal - 132 001, Haryana, India
| | - M K Singh
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR - National Dairy Research Institute, Karnal - 132 001, Haryana, India
| |
Collapse
|
|
21
|
Yang B, Zhou W, Jiao J, Nielsen JB, Mathis MR, Heydarpour M, Lettre G, Folkersen L, Prakash S, Schurmann C, Fritsche L, Farnum GA, Lin M, Othman M, Hornsby W, Driscoll A, Levasseur A, Thomas M, Farhat L, Dubé MP, Isselbacher EM, Franco-Cereceda A, Guo DC, Bottinger EP, Deeb GM, Booher A, Kheterpal S, Chen YE, Kang HM, Kitzman J, Cordell HJ, Keavney BD, Goodship JA, Ganesh SK, Abecasis G, Eagle KA, Boyle AP, Loos RJF, Eriksson P, Tardif JC, Brummett CM, Milewicz DM, Body SC, Willer CJ. Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve. Nat Commun 2017; 8:15481. [PMID: 28541271 PMCID: PMC5458508 DOI: 10.1038/ncomms15481] [Citation(s) in RCA: 83] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Accepted: 03/31/2017] [Indexed: 01/09/2023] Open
Abstract
Bicuspid aortic valve (BAV) is a heritable congenital heart defect and an important risk factor for valvulopathy and aortopathy. Here we report a genome-wide association scan of 466 BAV cases and 4,660 age, sex and ethnicity-matched controls with replication in up to 1,326 cases and 8,103 controls. We identify association with a noncoding variant 151 kb from the gene encoding the cardiac-specific transcription factor, GATA4, and near-significance for p.Ser377Gly in GATA4. GATA4 was interrupted by CRISPR-Cas9 in induced pluripotent stem cells from healthy donors. The disruption of GATA4 significantly impaired the transition from endothelial cells into mesenchymal cells, a critical step in heart valve development.
Collapse
Affiliation(s)
- Bo Yang
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Wei Zhou
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Jiao Jiao
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Jonas B. Nielsen
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Michael R. Mathis
- Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Mahyar Heydarpour
- Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Guillaume Lettre
- Montreal Heart Institute, Montreal, Quebec, Canada HIT 1C8
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada QC H3T 1J4
| | - Lasse Folkersen
- Cardiovascular Medicine Unit, Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm SE-171 76, Sweden
- Center for Biological Sequence Analysis, Technical University of Denmark, Copenhagen DK-2800, Denmark
| | - Siddharth Prakash
- Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, USA
| | - Claudia Schurmann
- The Charles Bronfman Institute for Personalized Medicine, The Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Lars Fritsche
- Department of Biostatistics, University of Michigan, Ann Arbor, Michigan 48109, USA
- Norwegian University of Science and Technology, Trondheim 7491, Norway
| | - Gregory A. Farnum
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Maoxuan Lin
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Mohammad Othman
- Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105, USA
| | - Whitney Hornsby
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Anisa Driscoll
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Alexandra Levasseur
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Marc Thomas
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Linda Farhat
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Marie-Pierre Dubé
- Montreal Heart Institute, Montreal, Quebec, Canada HIT 1C8
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada QC H3T 1J4
| | - Eric M. Isselbacher
- Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Anders Franco-Cereceda
- Cardiothoracic Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm SE-171 76, Sweden
| | - Dong-chuan Guo
- Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, USA
| | - Erwin P. Bottinger
- The Charles Bronfman Institute for Personalized Medicine, The Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - G. Michael Deeb
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Anna Booher
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Sachin Kheterpal
- Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Y. Eugene Chen
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Hyun Min Kang
- Department of Biostatistics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Jacob Kitzman
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Heather J. Cordell
- Institute of Genetic Medicine, Newcastle University, Newcastle Upon Tyne NE1 3BZ, UK
| | - Bernard D. Keavney
- Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK
- Manchester Heart Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WL, UK
| | - Judith A. Goodship
- Institute of Genetic Medicine, Newcastle University, Newcastle Upon Tyne NE1 3BZ, UK
| | - Santhi K. Ganesh
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Gonçalo Abecasis
- Department of Biostatistics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Kim A. Eagle
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Alan P. Boyle
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Ruth J. F. Loos
- The Charles Bronfman Institute for Personalized Medicine, The Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
- The Mindich Child Health Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Per Eriksson
- Cardiovascular Medicine Unit, Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm SE-171 76, Sweden
| | - Jean-Claude Tardif
- Montreal Heart Institute, Montreal, Quebec, Canada HIT 1C8
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada QC H3T 1J4
| | - Chad M. Brummett
- Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48109, USA
| | - Dianna M. Milewicz
- Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, USA
| | - Simon C. Body
- Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Cristen J. Willer
- Frankel Cardiovascular Center, University of Michigan, Ann Arbor, Michigan 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA
| |
Collapse
|
|
22
|
Neganova I, Chichagova V, Armstrong L, Lako M. A critical role for p38MAPK signalling pathway during reprogramming of human fibroblasts to iPSCs. Sci Rep 2017; 7:41693. [PMID: 28155868 PMCID: PMC5290526 DOI: 10.1038/srep41693] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 12/09/2016] [Indexed: 01/17/2023] Open
Abstract
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. Reprogramming is a stepwise process with well-defined stages of initiation, maturation and stabilisation which are critically dependent on interactions between key pluripotency transcription factors, epigenetic regulators and signalling pathways. In this manuscript we have investigated the role of p38 MAPK signalling pathway and have shown a subpopulation- and phase-specific pattern of activation occurring during the initiation and maturation stage of reprogramming in partially and fully reprogrammed cells respectively. Downregulation of p38 MAPK activity via RNA interference or small molecule inhibitor led to cell accumulation in G1 phase of the cell cycle and reduced expression of cell cycle regulators during the initiation stage of reprogramming. This was associated with a significant downregulation of key pluripotency marker expression, disruption of mesenchymal to epithelial transition (MET), increased expression of differentiation markers and presence of partially reprogrammed cells which retained a typical gene expression profile of mesendodermal cells and were unable to progress to fully reprogrammed phenotype. Together our data indicate an important role for p38 MAPK activity in proliferation, MET progression and establishment of pluripotent phenotype, which are necessary steps for the development of human iPSCs.
Collapse
Affiliation(s)
- Irina Neganova
- Institute of Genetic Medicine, Newcastle University, International Centre for Life, NE1 3BZ, UK
| | - Valeria Chichagova
- Institute of Genetic Medicine, Newcastle University, International Centre for Life, NE1 3BZ, UK
| | - Lyle Armstrong
- Institute of Genetic Medicine, Newcastle University, International Centre for Life, NE1 3BZ, UK
| | - Majlinda Lako
- Institute of Genetic Medicine, Newcastle University, International Centre for Life, NE1 3BZ, UK
| |
Collapse
|
|
23
|
Abstract
Induced pluripotent stem (iPS) cell reprogramming and direct reprogramming are promising approaches for disease modeling and personalized medicine. However, these processes are yet to be optimized. Biomaterials are increasingly integrated into cell reprogramming strategies in order to engineer the microenvironment, improve reprogramming efficiency and achieve effective in situ cell reprogramming. Although there are some studies on the role of biomaterials in iPS cell reprogramming, their effect on direct cell conversion has not been fully explored. Here we review the recent advances in the use of biomaterials for iPS cell reprogramming and direct reprogramming, with a focus on the biophysical aspect. We further highlight the future challenges and directions of the field.
Collapse
Affiliation(s)
- Sze Yue Wong
- Department of Bioengineering, University of California, Berkeley
| | - Jennifer Soto
- Department of Bioengineering, University of California, Berkeley
| | - Song Li
- Department of Bioengineering, University of California, Berkeley.,Department of Bioengineering and Department of Medicine, University of California, Los Angeles
| |
Collapse
|
|
24
|
Singh VP, Mathison M, Patel V, Sanagasetti D, Gibson BW, Yang J, Rosengart TK. MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1. J Am Heart Assoc 2016; 5:e003922. [PMID: 27930352 PMCID: PMC5210349 DOI: 10.1161/jaha.116.003922] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 09/23/2016] [Indexed: 01/14/2023]
Abstract
BACKGROUND Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. METHODS AND RESULTS Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. CONCLUSIONS These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming.
Collapse
Affiliation(s)
- Vivek P Singh
- Department of Surgery, Baylor College of Medicine, Houston, TX
| | - Megumi Mathison
- Department of Surgery, Baylor College of Medicine, Houston, TX
| | | | | | - Brian W Gibson
- Center for Comparative Medicine, Baylor College of Medicine, Houston, TX
| | - Jianchang Yang
- Department of Surgery, Baylor College of Medicine, Houston, TX
| | | |
Collapse
|
|
25
|
Differentiation defect in neural crest-derived smooth muscle cells in patients with aortopathy associated with bicuspid aortic valves. EBioMedicine 2016; 10:282-90. [PMID: 27394642 PMCID: PMC5006642 DOI: 10.1016/j.ebiom.2016.06.045] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 06/28/2016] [Accepted: 06/29/2016] [Indexed: 12/20/2022] Open
Abstract
Individuals with bicuspid aortic valves (BAV) are at a higher risk of developing thoracic aortic aneurysms (TAA) than patients with trileaflet aortic valves (TAV). The aneurysms associated with BAV most commonly involve the ascending aorta and spare the descending aorta. Smooth muscle cells (SMCs) in the ascending and descending aorta arise from neural crest (NC) and paraxial mesoderm (PM), respectively. We hypothesized defective differentiation of the neural crest stem cells (NCSCs)-derived SMCs but not paraxial mesoderm cells (PMCs)-derived SMCs contributes to the aortopathy associated with BAV. When induced pluripotent stem cells (iPSCs) from BAV/TAA patients were differentiated into NCSC-derived SMCs, these cells demonstrated significantly decreased expression of marker of SMC differentiation (MYH11) and impaired contraction compared to normal control. In contrast, the PMC-derived SMCs were similar to control cells in these aspects. The NCSC-SMCs from the BAV/TAA also showed decreased TGF-β signaling based on phosphorylation of SMAD2, and increased mTOR signaling. Inhibition of mTOR pathway using rapamycin rescued the aberrant differentiation. Our data demonstrates that decreased differentiation and contraction of patient's NCSC-derived SMCs may contribute to that aortopathy associated with BAV.
Model of the BAV/TAA is established using iPSCs differentiated into SMCs through distinct embryonic progenitors. BAV/TAA iPSCs differentiated SMCs from NCSCs were impaired in contractile function, whereas, iPSCs PMCs-SMCs were normal. NCSC-SMCs from BAV/TAA decreased in TGF-β signaling but increased in mTOR signaling. Rapamycin normalized contractile function. Aneurysms associated with bicuspid aortic valves (BAV) most commonly involve the ascending aorta and spare the descending aorta. Smooth muscle cells (SMCs) in the ascending and descending aorta arise from neural crest (NC) and paraxial mesoderm (PM), respectively. When induced pluripotent stem cells (iPSCs) from BAV patients were differentiated into NC stem cells (NCSCs)-derived SMCs, these cells demonstrated impaired contraction compared to normal control. In contrast, the PM cells-derived SMCs were similar to control cells. The NCSC-SMCs from the BAV/TAA also showed increased mTOR signaling. Inhibition of mTOR pathway using rapamycin rescued the aberrant differentiation.
Collapse
|
|
26
|
Yuen SM, Kwok HF. Temporal establishment of neural cell identity in vivo and in vitro. J Tissue Eng Regen Med 2016; 11:2582-2589. [PMID: 27061786 DOI: 10.1002/term.2158] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2015] [Accepted: 01/29/2016] [Indexed: 01/09/2023]
Abstract
Understanding cell fate specification is particularly useful because it enables biologists to generate specific neural cell types for treating currently untreatable neurological diseases. Traditionally, lineage-specific progenitors are generated in vitro from pluripotent cells, after which they may be channeled into more mature cell types in a stage-specific manner, which is similar to the way cells behave during development. However, the emergence of induced pluripotent stem cells means that specific cell types can be generated directly from fibroblasts or other somatic cell types, thus bypassing all of the necessary steps that happen in vivo. Based on this information, the present review first explores the regulatory circuitry that drives cell fate specification over time in vivo. In particular, it describes how the appearance of specific neuronal and glial cell types is governed by an intrinsic biological clock, followed by a discussion of how this can be achieved through the temporal expression of intracellular regulators in relation to cell-specific Dnase I hypersensitivity sites, promoters and enhancers. Cell fate acquisition in vitro was then examined in an attempt to evaluate whether the temporal regulation neural cell fate in vivo is still relevant to the generation of reprogrammed neural stem cells and neurons. Copyright © 2016 John Wiley & Sons, Ltd.
Collapse
Affiliation(s)
- Shun Ming Yuen
- Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, SAR, China
| | - Hang Fai Kwok
- Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, SAR, China
| |
Collapse
|
|
27
|
Chen J, Gao Y, Huang H, Xu K, Chen X, Jiang Y, Li H, Gao S, Tao Y, Wang H, Zhang Y, Wang H, Cai T, Gao S. The combination of Tet1 with Oct4 generates high-quality mouse-induced pluripotent stem cells. Stem Cells 2015; 33:686-98. [PMID: 25331067 DOI: 10.1002/stem.1879] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2014] [Revised: 09/02/2014] [Accepted: 09/04/2014] [Indexed: 01/20/2023]
Abstract
The DNA dioxygenase Tet1 has recently been proposed to play an important role in the reprogramming of somatic cells to pluripotency. Its oxidization product 5-hydroxymethylcytosine, formerly considered an intermediate in the demethylation of 5-methylcytosine, has recently been implicated as being important in epigenetic reprogramming. Here, we provide evidence that Tet1 (T) can replace multiple transcription factors during somatic cell reprogramming and can generate high-quality mouse induced pluripotent stem cells (iPSCs) with Oct4 (O). The OT-iPSCs can efficiently produce viable mice derived entirely from iPSCs through tetraploid complementation; all 47 adult OT-iPSC mice grew healthily, without tumorigenesis, and had a normal life span. Furthermore, a new secondary reprogramming system was established using the OT all-iPSC mice-derived somatic cells. Our results provide the first evidence that the DNA dioxygenase Tet1 can replace multiple pluripotency transcription factors and can generate high-quality iPSCs with Oct4.
Collapse
Affiliation(s)
- Jiayu Chen
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, People's Republic of China; National Institute of Biological Sciences, NIBS, Beijing, People's Republic of China
| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Jiang Y, Du M, Wu M, Zhu Y, Zhao X, Cao X, Li X, Long P, Li W, Hu B. Phosphatidic Acid Improves Reprogramming to Pluripotency by Reducing Apoptosis. Stem Cells Dev 2015; 25:43-54. [PMID: 26451619 DOI: 10.1089/scd.2015.0159] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Generation of induced pluripotent stem cells (iPSCs) requires a considerable amount of lipids, such as phosphatidic acid (PA), to meet the needs of subsequent rapid cell division and proliferation. However, it is unclear whether PA, a biosynthetic precursor of lipids, affects reprogramming. By using lentiviral expression of the Yamanaka factors in mouse embryonic fibroblasts for reprogramming, we identified that PA is beneficial for the generation of iPS colonies. Inhibiting the generation of cellular PA dramatically decreased the number of iPSCs. Consistently, 400 μM PA improved iPSC generation by more than 4- to 5-fold. iPSCs generated in the presence of PA (PA-iPS) expressed pluripotent markers such as Oct4 and Nanog, differentiated into cells of the three germ layers in vitro, and contributed to chimeric mice when injected into blastocysts. The improved efficiency was primarily due to reduction of apoptosis as sufficient PA increased the accumulation of cardiolipin in the inner membrane of the mitochondria, which reduced the release of cytochrome c and, in turn, suppressed apoptosis by inhibiting caspase-7. The relatively higher amount of Bcl-2 in PA treatment also inhibited apoptosis. In addition, an accompanied sequential change from epithelial-to-mesenchymal transition (EMT) at the initial phase of reprogramming to mesenchymal-to-epithelial transition (MET) was also detected. Our microarray data, which also supported our results, indicated the presence of significant membrane enrichment genes, thus suggesting that PA may function through membrane-anchored proteins. We thus identified a novel type of culture supplement that improves the efficiency of reprogramming and could be valuable for the generation of high-quality iPS cells.
Collapse
Affiliation(s)
- Yuan Jiang
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Mingxia Du
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,3 School of Life Sciences, Anhui University , Hefei, China
| | - Menghua Wu
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Yanbing Zhu
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Xing Zhao
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Xu Cao
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Xin Li
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Peipei Long
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Wei Li
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| | - Baoyang Hu
- 1 The State Key Laboratory of Reproductive Biology, Institute of Zoology , Chinese Academy of Sciences, Beijing, China .,2 University of Chinese Academy of Sciences , Beijing, China
| |
Collapse
|
|
29
|
Chen Q, Gao S, He W, Kou X, Zhao Y, Wang H, Gao S. Xist repression shows time-dependent effects on the reprogramming of female somatic cells to induced pluripotent stem cells. Stem Cells 2015; 32:2642-56. [PMID: 24965076 DOI: 10.1002/stem.1775] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2013] [Revised: 05/17/2014] [Accepted: 05/23/2014] [Indexed: 11/10/2022]
Abstract
Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X-inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal-to-epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre-iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre-iPSCs to iPSCs.
Collapse
Affiliation(s)
- Qi Chen
- School of Life Sciences, Beijing Normal University, Beijing, China; National Institute of Biological Sciences (NIBS), Beijing, China
| | | | | | | | | | | | | |
Collapse
|
|
30
|
Mu WL, Wang YJ, Xu P, Hao DL, Liu XZ, Wang TT, Chen F, Chen HZ, Lv X, Liu DP. Sox2 Deacetylation by Sirt1 Is Involved in Mouse Somatic Reprogramming. Stem Cells 2015; 33:2135-47. [DOI: 10.1002/stem.2012] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2014] [Accepted: 03/02/2015] [Indexed: 11/09/2022]
Affiliation(s)
- Wen-Li Mu
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Ya-Jun Wang
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Peng Xu
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - De-Long Hao
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Xiu-Zhen Liu
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Ting-Ting Wang
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Feng Chen
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Hou-Zao Chen
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - Xiang Lv
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| | - De-Pei Liu
- Department of Biochemistry and Molecular Biology; State Key Laboratory of Medical Molecular Biology; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing People's Republic of China
| |
Collapse
|
|
31
|
Tan F, Qian C, Tang K, Abd-Allah SM, Jing N. Inhibition of transforming growth factor β (TGF-β) signaling can substitute for Oct4 protein in reprogramming and maintain pluripotency. J Biol Chem 2014; 290:4500-11. [PMID: 25548277 DOI: 10.1074/jbc.m114.609016] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/β-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-β signaling in this process remains elusive. Here we show that inhibiting TGF-β signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-β signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-β signaling inhibition in the generation and maintenance of PSCs.
Collapse
Affiliation(s)
- Fangzhi Tan
- From the State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Cheng Qian
- From the State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Ke Tang
- the Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, China, and
| | - Saber Mohamed Abd-Allah
- the Theriogenology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt
| | - Naihe Jing
- From the State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China,
| |
Collapse
|
|
32
|
Li Z, Dang J, Chang KY, Rana TM. MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming. RNA (NEW YORK, N.Y.) 2014; 20:1900-1915. [PMID: 25336587 PMCID: PMC4238355 DOI: 10.1261/rna.043745.113] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 08/27/2014] [Indexed: 06/04/2023]
Abstract
Somatic cells can be reprogrammed to reach an embryonic stem cell-like state by overexpression of defined factors. Recent studies have greatly improved the efficiency of the reprogramming process but the underlying mechanisms regulating the transition from a somatic to a pluripotent state are still relatively unknown. MicroRNAs (miRs) are small noncoding RNAs that primarily regulate target gene expression post-transcriptionally. Here we present a systematic and comprehensive study of microRNAs in mouse embryonic fibroblasts (MEFs) during the early stage of cell fate decisions and reprogramming to a pluripotent state, in which significant transcriptional and epigenetic changes occur. One microRNA found to be highly induced during this stage of reprogramming, miR-135b, targeted the expression of extracellular matrix (ECM) genes including Wisp1 and Igfbp5. Wisp1 was shown to be a key regulator of additional ECM genes that serve as barriers to reprogramming. Regulation of Wisp 1 is likely mediated through biglycan, a glycoprotein highly expressed in MEFs that is silenced in reprogrammed cells. Collectively, this report reveals a novel link between microRNA-mediated regulation of ECM formation and somatic cell reprogramming, and demonstrates that microRNAs are powerful tools to dissect the intracellular and extracellular molecular mechanisms of reprogramming.
Collapse
Affiliation(s)
- Zhonghan Li
- Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA
| | - Jason Dang
- Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA
| | - Kung-Yen Chang
- Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, California 92093, USA
| | - Tariq M Rana
- Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, California 92093, USA
| |
Collapse
|
|
33
|
Hawkins K, Joy S, McKay T. Cell signalling pathways underlying induced pluripotent stem cell reprogramming. World J Stem Cells 2014; 6:620-628. [PMID: 25426259 PMCID: PMC4178262 DOI: 10.4252/wjsc.v6.i5.620] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2014] [Revised: 09/03/2014] [Accepted: 09/17/2014] [Indexed: 02/07/2023] Open
Abstract
Induced pluripotent stem (iPS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and cMyc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few “stabilisation competent” cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-iPS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, iPS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As iPS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.
Collapse
|
|
34
|
Rubert M, Dehli J, Li YF, Taskin MB, Xu R, Besenbacher F, Chen M. Electrospun PCL/PEO coaxial fibers for basic fibroblast growth factor delivery. J Mater Chem B 2014; 2:8538-8546. [DOI: 10.1039/c4tb01258e] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
|
|
35
|
Qin H, Diaz A, Blouin L, Lebbink RJ, Patena W, Tanbun P, LeProust EM, McManus MT, Song JS, Ramalho-Santos M. Systematic identification of barriers to human iPSC generation. Cell 2014; 158:449-461. [PMID: 25036638 DOI: 10.1016/j.cell.2014.05.040] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Revised: 03/05/2014] [Accepted: 05/27/2014] [Indexed: 12/13/2022]
Abstract
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. To elucidate endogenous barriers limiting this process, we systematically dissected human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation, and mechanistic investigation of relevant pathways and networks. We identify reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins inhibit reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGF-β signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel, or feedforward loop architectures to antagonize reprogramming. These results provide a global view of barriers to human cellular reprogramming.
Collapse
Affiliation(s)
- Han Qin
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Departments of Ob/Gyn and Pathology, Center for Reproductive Sciences, and Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Aaron Diaz
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Laure Blouin
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Departments of Ob/Gyn and Pathology, Center for Reproductive Sciences, and Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Robert Jan Lebbink
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, Diabetes Center, and the WM Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Weronika Patena
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, Diabetes Center, and the WM Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Priscilia Tanbun
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Departments of Ob/Gyn and Pathology, Center for Reproductive Sciences, and Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Emily M LeProust
- Genomics Solution Unit, Agilent Technologies Inc., Santa Clara, CA 95051, USA
| | - Michael T McManus
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, Diabetes Center, and the WM Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Jun S Song
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Epidemiology and Biostatistics and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.
| | - Miguel Ramalho-Santos
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Departments of Ob/Gyn and Pathology, Center for Reproductive Sciences, and Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
| |
Collapse
|
|
36
|
Liu K, Wang F, Ye X, Wang L, Yang J, Zhang J, Liu L. KSR-based medium improves the generation of high-quality mouse iPS cells. PLoS One 2014; 9:e105309. [PMID: 25171101 PMCID: PMC4149410 DOI: 10.1371/journal.pone.0105309] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 07/23/2014] [Indexed: 02/05/2023] Open
Abstract
Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP+ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP+ iPS cells. Remarkably, PD can rescue Nanog-GFP+ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells.
Collapse
Affiliation(s)
- Kai Liu
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Fang Wang
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Xiaoying Ye
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Lingling Wang
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Jiao Yang
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Jingzhuo Zhang
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Lin Liu
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China
- * E-mail:
| |
Collapse
|
|
37
|
Ye S, Liu D, Ying QL. Signaling pathways in induced naïve pluripotency. Curr Opin Genet Dev 2014; 28:10-5. [PMID: 25173148 DOI: 10.1016/j.gde.2014.08.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2014] [Revised: 06/29/2014] [Accepted: 08/08/2014] [Indexed: 10/24/2022]
Abstract
Pluripotent stem cells have become powerful tools for both research and regenerative medicine. To date, however, only mouse and rat embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) have the ability to contribute to the formation of germline-competent chimeras. These stem cells are thus considered as 'naïve' pluripotent stem cells. Several signaling pathways have been identified to play a critical role in the induction and maintenance of this naïve pluripotent state. Understanding how these pathways induce and maintain naïve pluripotency will likely lead to the generation of germline-competent naïve ESCs/iPSCs from humans and animals phylogenetically close to humans.
Collapse
Affiliation(s)
- Shoudong Ye
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, PR China; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Dahai Liu
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, PR China
| | - Qi-Long Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
| |
Collapse
|
|
38
|
Kim JS, Choi HW, Choi S, Seo HG, Moon SH, Chung HM, Do JT. Conversion of partially reprogrammed cells to fully pluripotent stem cells is associated with further activation of stem cell maintenance- and gamete generation-related genes. Stem Cells Dev 2014; 23:2637-48. [PMID: 24892478 DOI: 10.1089/scd.2014.0020] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.
Collapse
Affiliation(s)
- Jong Soo Kim
- 1 Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University , Seoul, Republic of Korea
| | | | | | | | | | | | | |
Collapse
|
|
39
|
Cordie T, Harkness T, Jing X, Carlson-Stevermer J, Mi HY, Turng LS, Saha K. Nanofibrous Electrospun Polymers for Reprogramming Human Cells. Cell Mol Bioeng 2014. [DOI: 10.1007/s12195-014-0341-z] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
|
|
40
|
Xu Y, Liu L, Laslett AL, Esteban MA. Cell reprogramming: Into the groove. NATURE MATERIALS 2013; 12:1082-1084. [PMID: 24257131 DOI: 10.1038/nmat3821] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Affiliation(s)
- Yan Xu
- Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and the Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou 510530, China
| | | | | | | |
Collapse
|
|
41
|
Teng S, Liu C, Krettek C, Jagodzinski M. The application of induced pluripotent stem cells for bone regeneration: current progress and prospects. TISSUE ENGINEERING PART B-REVIEWS 2013; 20:328-39. [PMID: 24102431 DOI: 10.1089/ten.teb.2013.0301] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Loss of healthy bone tissue and dysosteogenesis are still common and significant problems in clinics. Cell-based therapy using mesenchymal stem cells (MSCs) has been performed in patients for quite some time, but the inherent drawbacks of these cells, such as the reductions in proliferation rate and osteogenic differentiation potential that occur with aging, greatly limit their further application. Moreover, embryonic stem cells (ESCs) have brought new hope to osteoregenerative medicine because of their full pluripotent differentiation potential and excellent performance in bone regeneration. However, the ethical issues involved in destroying human embryos and the immune reactions that occur after transplantation are two major stumbling blocks impeding the clinical application of ESCs. Instead, induced pluripotent stem cells (iPSCs), which are ESC-like pluripotent cells that are reprogrammed from adult somatic cells using defined transcription factors, are considered a more promising source of cells for regenerative medicine because they present no ethical or immunological issues. Here, we summarize the primary technologies for generating iPSCs and the biological properties of these cells, review the current advances in iPSC-based bone regeneration and, finally, discuss the remaining challenges associated with these cells, particularly safety issues and their potential application for osteoregenerative medicine.
Collapse
Affiliation(s)
- Songsong Teng
- 1 Department of Orthopedic Trauma, Hanover Medical School (MHH) , Hanover, Germany
| | | | | | | |
Collapse
|