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Chen Y, Shao Y. Stem Cell-Based Embryo Models: En Route to a Programmable Future. J Mol Biol 2021; 434:167353. [PMID: 34774563 DOI: 10.1016/j.jmb.2021.167353] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Revised: 11/04/2021] [Accepted: 11/04/2021] [Indexed: 01/10/2023]
Abstract
Early-stage human embryogenesis, such as implantation, gastrulation, and neurulation, are critical for successful pregnancy. For decades, our knowledge about these stages has been limited by the inaccessibility to such embryo specimens in vivo and the difficulty in rebuilding them in vitro. Although human embryos could be cultured in vitro beyond implantation, it remains challenging for the cultured embryos to recapitulate the continuous, coordinated morphogenesis and cytodifferentiation as seen in vivo. Stem cell-based embryo models, mainly derived from human pluripotent stem cells, are organized structures mimicking essential developmental processes in the early-stage human embryos. Despite their invaluable potentials, most embryo models are based on the self-organization of human pluripotent stem cells, which are limited in controllability, reproducibility, and developmental fidelity. Recently, the integration of bioengineered tools and stem cell biology has fueled a technological transformation towards programmable, highly complex, high-fidelity stem cell-based embryo models. Given its scientific and clinical significance, we present an overview of recent paradigm-shifting advances as well as historical perspectives regarding the past, present, and future of synthetic human embryology. Following the developmental roadmap of human embryogenesis, we critically review existing stem cell-based models for implantation, gastrulation, and neurulation, respectively. We highlight the limitations encountered by autonomous self-organization strategy and discuss the concept and application of guided cell organization as a game-changer for innovating next-generation embryo models. Future endeavors in synthetic human embryology should rationally leverage both the self-organizing power and programmable microenvironmental guidance to secure faithful reconstructions of the hierarchical orders of human embryogenesis in vitro.
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Affiliation(s)
- Yunping Chen
- Institute of Biomechanics and Medical Engineering, Applied Mechanics Laboratory, Department of Engineering Mechanics, Tsinghua University, Beijing 100084, China
| | - Yue Shao
- Institute of Biomechanics and Medical Engineering, Applied Mechanics Laboratory, Department of Engineering Mechanics, Tsinghua University, Beijing 100084, China.
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Lezmi E, Weissbein U, Golan-Lev T, Nissim-Rafinia M, Meshorer E, Benvenisty N. The Chromatin Regulator ZMYM2 Restricts Human Pluripotent Stem Cell Growth and Is Essential for Teratoma Formation. Stem Cell Reports 2020; 15:1275-1286. [PMID: 32559458 PMCID: PMC7724477 DOI: 10.1016/j.stemcr.2020.05.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2019] [Revised: 05/19/2020] [Accepted: 05/19/2020] [Indexed: 01/13/2023] Open
Abstract
Chromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.
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Affiliation(s)
- Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, The Hebrew University, Jerusalem, Israel
| | - Uri Weissbein
- The Azrieli Center for Stem Cells and Genetic Research, The Hebrew University, Jerusalem, Israel
| | - Tamar Golan-Lev
- The Azrieli Center for Stem Cells and Genetic Research, The Hebrew University, Jerusalem, Israel; Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel
| | - Malka Nissim-Rafinia
- Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel; Edmond and Lily Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem, Israel
| | - Eran Meshorer
- Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel; Edmond and Lily Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem, Israel.
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, The Hebrew University, Jerusalem, Israel; Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.
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Minn KT, Fu YC, He S, Dietmann S, George SC, Anastasio MA, Morris SA, Solnica-Krezel L. High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures. eLife 2020. [PMID: 33206048 DOI: 10.1101/2020.1101.1122.915777] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/15/2023] Open
Abstract
During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement.
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Affiliation(s)
- Kyaw Thu Minn
- Department of Biomedical Engineering, Washington University, St. Louis, United States
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States
| | - Yuheng C Fu
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States
- Department of Genetics, Washington University School of Medicine, St. Louis, United States
- Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, United States
| | - Shenghua He
- Department of Computer Science & Engineering, Washington University, St. Louis, United States
| | - Sabine Dietmann
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States
- Division of Nephrology, Washington University School of Medicine, St. Louis, United States
- Institute for Informatics, Washington University School of Medicine, St. Louis, United States
| | - Steven C George
- Department of Biomedical Engineering, University of California, Davis, Davis, United States
| | - Mark A Anastasio
- Department of Biomedical Engineering, Washington University, St. Louis, United States
- Department of Bioengineering, University of Illinois, Urbana-Champaign, United States
| | - Samantha A Morris
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States
- Department of Genetics, Washington University School of Medicine, St. Louis, United States
- Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, United States
| | - Lilianna Solnica-Krezel
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States
- Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, United States
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Minn KT, Fu YC, He S, Dietmann S, George SC, Anastasio MA, Morris SA, Solnica-Krezel L. High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures. eLife 2020; 9:e59445. [PMID: 33206048 PMCID: PMC7728446 DOI: 10.7554/elife.59445] [Citation(s) in RCA: 69] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 11/17/2020] [Indexed: 12/29/2022] Open
Abstract
During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement.
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Affiliation(s)
- Kyaw Thu Minn
- Department of Biomedical Engineering, Washington UniversitySt. LouisUnited States
- Department of Developmental Biology, Washington University School of MedicineSt. LouisUnited States
| | - Yuheng C Fu
- Department of Developmental Biology, Washington University School of MedicineSt. LouisUnited States
- Department of Genetics, Washington University School of MedicineSt. LouisUnited States
- Center of Regenerative Medicine, Washington University School of MedicineSt. LouisUnited States
| | - Shenghua He
- Department of Computer Science & Engineering, Washington UniversitySt. LouisUnited States
| | - Sabine Dietmann
- Department of Developmental Biology, Washington University School of MedicineSt. LouisUnited States
- Division of Nephrology, Washington University School of MedicineSt. LouisUnited States
- Institute for Informatics, Washington University School of MedicineSt. LouisUnited States
| | - Steven C George
- Department of Biomedical Engineering, University of California, DavisDavisUnited States
| | - Mark A Anastasio
- Department of Biomedical Engineering, Washington UniversitySt. LouisUnited States
- Department of Bioengineering, University of IllinoisUrbana-ChampaignUnited States
| | - Samantha A Morris
- Department of Developmental Biology, Washington University School of MedicineSt. LouisUnited States
- Department of Genetics, Washington University School of MedicineSt. LouisUnited States
- Center of Regenerative Medicine, Washington University School of MedicineSt. LouisUnited States
| | - Lilianna Solnica-Krezel
- Department of Developmental Biology, Washington University School of MedicineSt. LouisUnited States
- Center of Regenerative Medicine, Washington University School of MedicineSt. LouisUnited States
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Zeevaert K, Elsafi Mabrouk MH, Wagner W, Goetzke R. Cell Mechanics in Embryoid Bodies. Cells 2020; 9:E2270. [PMID: 33050550 PMCID: PMC7599659 DOI: 10.3390/cells9102270] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 10/06/2020] [Accepted: 10/09/2020] [Indexed: 12/14/2022] Open
Abstract
Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell-cell interactions and lumen formation-a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization-e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions.
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Affiliation(s)
- Kira Zeevaert
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Mohamed H. Elsafi Mabrouk
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Wolfgang Wagner
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Roman Goetzke
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
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Song Y, Lim JY, Lim T, Im KI, Kim N, Nam YS, Jeon YW, Shin JC, Ko HS, Park IY, Cho SG. Human mesenchymal stem cells derived from umbilical cord and bone marrow exert immunomodulatory effects in different mechanisms. World J Stem Cells 2020; 12:1032-1049. [PMID: 33033563 PMCID: PMC7524695 DOI: 10.4252/wjsc.v12.i9.1032] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2020] [Revised: 06/20/2020] [Accepted: 07/19/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) are an attractive tool to treat graft-versus-host disease because of their unique immunoregulatory properties. Although human bone marrow-derived MSCs (BM-MSCs) were the most widely used MSCs in cell therapy until recently, MSCs derived from human umbilical cords (UC-MSCs) have gained popularity as cell therapy material for their ethical and noninvasive collection.
AIM To investigate the difference in mechanisms of the immunosuppressive effects of UC-MSCs and BM-MSCs.
METHODS To analyze soluble factors expressed by MSCs, such as indolamine 2,3-dioxygenase, cyclooxygenase-2, prostaglandin E2 and interleukin (IL)-6, inflammatory environments in vitro were reconstituted with combinations of interferon-gamma (IFN-γ), tumor necrosis factor alpha and IL-1β or with IFN-γ alone. Activated T cells were cocultured with MSCs treated with indomethacin and/or anti-IL-10. To assess the ability of MSCs to inhibit T helper 17 cells and induce regulatory T cells, induced T helper 17 cells were cocultured with MSCs treated with indomethacin or anti-IL-10. Xenogeneic graft-versus-host disease was induced in NOG mice (NOD/Shi-scid/IL-2Rγnull) and UC-MSCs or BM-MSCs were treated as cell therapies.
RESULTS Our data demonstrated that BM-MSCs and UC-MSCs shared similar phenotypic characteristics and immunomodulation abilities. BM-MSCs expressed more indolamine 2,3-dioxygenase after cytokine stimulation with different combinations of IFN-γ, tumor necrosis factor alpha-α and IL-1β or IFN-γ alone. UC-MSCs expressed more prostaglandin E2, IL-6, programmed death-ligand 1 and 2 in the in vitro inflammatory environment. Cyclooxygenase-2 and IL-10 were key factors in the immunomodulatory mechanisms of both MSCs. In addition, UC-MSCs inhibited more T helper 17 cells and induced more regulatory T cells than BM-MSCs. UC-MSCs and BM-MSCs exhibited similar effects on attenuating graft-versus-host disease.
CONCLUSION UC-MSCs and BM-MSCs exert similar immunosuppressive effects with different mechanisms involved. These findings suggest that UC-MSCs have distinct immunoregulatory functions and may substitute BM-MBSCs in the field of cell therapy.
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Affiliation(s)
- Yunejin Song
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Department of Biomedicine and Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Jung-Yeon Lim
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
| | - Taekyu Lim
- Division of Hematology Oncology, Department of Internal Medicine, Veterans Health Service Medical Center, Seoul 05368, South Korea
| | - Keon-Il Im
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Nayoun Kim
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Young-Sun Nam
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Young-Woo Jeon
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Jong Chul Shin
- Department of Obstetrics and Gynecology, CHA Bundang Medical Center, CHA University, Seongnam 13496, South Korea
| | - Hyun Sun Ko
- Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - In Yang Park
- Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
| | - Seok-Goo Cho
- Institute for Translational Research and Molecular Imaging, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
- Division of Hematology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea
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Sart S, Bejoy J, Li Y. Characterization of 3D pluripotent stem cell aggregates and the impact of their properties on bioprocessing. Process Biochem 2017. [DOI: 10.1016/j.procbio.2016.05.024] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid. ZYGOTE 2017; 25:341-357. [DOI: 10.1017/s0967199417000211] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
SummaryThis study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.
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Avior Y, Lezmi E, Yanuka D, Benvenisty N. Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells. Stem Cell Reports 2017; 8:1354-1365. [PMID: 28392220 PMCID: PMC5425613 DOI: 10.1016/j.stemcr.2017.03.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2016] [Revised: 03/06/2017] [Accepted: 03/07/2017] [Indexed: 12/18/2022] Open
Abstract
Human embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1−/− hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1−/− cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1–/–-specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1.
RB1-null hESCs were generated using CRISPR/Cas9 RB1−/− hESCs generate large, neural-enriched teratomas, possibly by ZEB1 activation RB1 inactivation triggers aberrant mitochondrial abundance and function Unbiased drug screening found that carboplatin specifically targets RB1-null cells
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Affiliation(s)
- Yishai Avior
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Dorit Yanuka
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel.
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Kandasamy M, Roll L, Langenstroth D, Brüstle O, Faissner A. Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs). Cell Tissue Res 2017; 368:531-549. [DOI: 10.1007/s00441-017-2594-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Accepted: 02/23/2017] [Indexed: 12/20/2022]
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Kaitsuka T, Kobayashi K, Otsuka W, Kubo T, Hakim F, Wei FY, Shiraki N, Kume S, Tomizawa K. Erythropoietin facilitates definitive endodermal differentiation of mouse embryonic stem cells via activation of ERK signaling. Am J Physiol Cell Physiol 2017; 312:C573-C582. [PMID: 28298334 DOI: 10.1152/ajpcell.00071.2016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Revised: 03/06/2017] [Accepted: 03/06/2017] [Indexed: 01/07/2023]
Abstract
Artificially generated pancreatic β-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic β-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and Akt. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic β-cells.
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Affiliation(s)
- Taku Kaitsuka
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kohei Kobayashi
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Wakako Otsuka
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takuya Kubo
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Farzana Hakim
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Fan-Yan Wei
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Nobuaki Shiraki
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; and.,Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
| | - Shoen Kume
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; and.,Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
| | - Kazuhito Tomizawa
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan;
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Wen J, Zeng Y, Fang Z, Gu J, Ge L, Tang F, Qu Z, Hu J, Cui Y, Zhang K, Wang J, Li S, Sun Y, Jin Y. Single-cell analysis reveals lineage segregation in early post-implantation mouse embryos. J Biol Chem 2017; 292:9840-9854. [PMID: 28298438 DOI: 10.1074/jbc.m117.780585] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Revised: 03/13/2017] [Indexed: 11/06/2022] Open
Abstract
The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. In particular, the individual definitive endoderm (DE) cells have not been characterized in vivo Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (FGF) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the premesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6 Analysis of single-cell RNA-sequence data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.
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Affiliation(s)
- Jing Wen
- From the Key Laboratory of Stem Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine, Shanghai 200031
| | - Yanwu Zeng
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Zhuoqing Fang
- From the Key Laboratory of Stem Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine, Shanghai 200031
| | - Junjie Gu
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Laixiang Ge
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Fan Tang
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Zepeng Qu
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Jing Hu
- the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
| | - Yaru Cui
- the Stem Cell Translational Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China
| | - Kushan Zhang
- the Stem Cell Translational Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China
| | - Junbang Wang
- the Stem Cell Translational Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China
| | - Siguang Li
- the Stem Cell Translational Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China
| | - Yi Sun
- the Stem Cell Translational Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China
| | - Ying Jin
- From the Key Laboratory of Stem Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine, Shanghai 200031, .,the Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, and
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TeratoScore: Assessing the Differentiation Potential of Human Pluripotent Stem Cells by Quantitative Expression Analysis of Teratomas. Stem Cell Reports 2016; 4:967-74. [PMID: 26070610 PMCID: PMC4471824 DOI: 10.1016/j.stemcr.2015.05.006] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Revised: 05/06/2015] [Accepted: 05/06/2015] [Indexed: 12/22/2022] Open
Abstract
Teratoma formation is the gold standard assay for testing the capacity of human pluripotent stem cells to differentiate into all embryonic germ layers. Although widely used, little effort has been made to transform this qualitative assay into a quantitative one. Using gene expression data from a wide variety of cells, we created a scorecard representing tissues from all germ layers and extraembryonic tissues. TeratoScore, an online, open-source platform based on this scorecard, distinguishes pluripotent stem cell-derived teratomas from malignant tumors, translating cell potency into a quantitative measure (http://benvenisty.huji.ac.il/teratoscore.php). The teratomas used for the algorithm also allowed us to examine gene expression differences between tumors with a diploid karyotype and those initiated by aneuploid cells. Chromosomally aberrant teratomas show a significantly different gene expression signature from that of teratomas originating from diploid cells, particularly in central nervous system-specific genes, congruent with human chromosomal syndromes.
A gene scorecard representing human tissues from all germ layers was created A quantitative pluripotency test named TeratoScore was based on this scorecard TeratoScore distinguishes pluripotent stem cell-derived teratomas from other tumors Teratomas derived from aneuploid cells show aberrant tissue expression distribution
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High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling. PLoS One 2016; 11:e0148819. [PMID: 26859149 PMCID: PMC4747557 DOI: 10.1371/journal.pone.0148819] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2015] [Accepted: 01/22/2016] [Indexed: 02/02/2023] Open
Abstract
Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.
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15
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Son MY, Kwak JE, Kim YD, Cho YS. Proteomic and network analysis of proteins regulated by REX1 in human embryonic stem cells. Proteomics 2015; 15:2220-9. [PMID: 25736782 DOI: 10.1002/pmic.201400510] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Revised: 01/19/2015] [Accepted: 02/26/2015] [Indexed: 01/09/2023]
Abstract
Recent studies have suggested that REX1 (reduced expression 1) plays an important role in pluripotency, proliferation, and differentiation. However, the molecular mechanisms involved in REX1-dependent regulation of diverse cellular processes remain unclear. To elucidate the regulatory functions of REX1 in human embryonic stem cells (hESCs), comparative proteomic analysis was performed on REX1 RNAi specifically silenced hESCs. Analysis of the proteome via nano-LC-MS/MS identified 140 differentially expressed proteins (DEPs) displaying a >2-fold difference in expression level between control and REX1 knockdown (KD) hESCs, which were then compared with transcriptome data and validated by quantitative real-time RT-PCR and Western blotting. These DEPs were analyzed by GO, pathway, and functional clustering analyses to determine the molecular functions of the proteins and pathways regulated by REX1. The REX1 KD-mediated DEPs mapped to major biological processes involved in the regulation of ribosome-mediated translation and mitochondrial function. Functional network analysis revealed a highly interconnected network among these DEPs and indicated that these interconnected proteins are predominantly involved in translation and the regulation of mitochondrial organization. These findings regarding REX1-mediated regulatory network have revealed the contributions of REX1 to maintaining the status of hESCs and have improved our understanding of the molecular events that underlie the fundamental properties of hESCs.
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Affiliation(s)
- Mi-Young Son
- Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahangno, Yuseong-gu, Daejeon, Republic of Korea.,Department of functional genomics, University of Science & Technology, Gajungro, Yuseong-gu, Daejeon, Republic of Korea
| | - Jae Eun Kwak
- Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahangno, Yuseong-gu, Daejeon, Republic of Korea
| | - Young-Dae Kim
- Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahangno, Yuseong-gu, Daejeon, Republic of Korea
| | - Yee Sook Cho
- Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahangno, Yuseong-gu, Daejeon, Republic of Korea.,Department of functional genomics, University of Science & Technology, Gajungro, Yuseong-gu, Daejeon, Republic of Korea
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16
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Yu DX, Di Giorgio FP, Yao J, Marchetto MC, Brennand K, Wright R, Mei A, McHenry L, Lisuk D, Grasmick JM, Silberman P, Silberman G, Jappelli R, Gage FH. Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Reports 2014; 2:295-310. [PMID: 24672753 PMCID: PMC3964286 DOI: 10.1016/j.stemcr.2014.01.009] [Citation(s) in RCA: 209] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2013] [Revised: 01/17/2014] [Accepted: 01/20/2014] [Indexed: 02/07/2023] Open
Abstract
The availability of human pluripotent stem cells (hPSCs) offers the opportunity to generate lineage-specific cells to investigate mechanisms of human diseases specific to brain regions. Here, we report a differentiation paradigm for hPSCs that enriches for hippocampal dentate gyrus (DG) granule neurons. This differentiation paradigm recapitulates the expression patterns of key developmental genes during hippocampal neurogenesis, exhibits characteristics of neuronal network maturation, and produces PROX1+ neurons that functionally integrate into the DG. Because hippocampal neurogenesis has been implicated in schizophrenia (SCZD), we applied our protocol to SCZD patient-derived human induced pluripotent stem cells (hiPSCs). We found deficits in the generation of DG granule neurons from SCZD hiPSC-derived hippocampal NPCs with lowered levels of NEUROD1, PROX1, and TBR1, reduced neuronal activity, and reduced levels of spontaneous neurotransmitter release. Our approach offers important insights into the neurodevelopmental aspects of SCZD and may be a promising tool for drug screening and personalized medicine.
Hippocampal neurogenesis is modeled using human pluripotent stem cells Differentiated DG neurons are detected using lentiviral PROX1-GFP reporter construct Differentiated granule neurons functionally integrate into the dentate gyrus in vivo SCZD hiPSC-derived hippocampal NPCs present deficits in hippocampal neurogenesis
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Affiliation(s)
- Diana Xuan Yu
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Francesco Paolo Di Giorgio
- Neuroscience Discovery, Novartis Pharma AG, Novartis Institute for Biomedical Research, Postfach, Basel CH-4002, Switzerland
| | - Jun Yao
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Maria Carolina Marchetto
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Kristen Brennand
- Department of Neuroscience/Psychiatry, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10059, USA
| | - Rebecca Wright
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Arianna Mei
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Lauren McHenry
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - David Lisuk
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Jaeson Michael Grasmick
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Pedro Silberman
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Giovanna Silberman
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Roberto Jappelli
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Fred H Gage
- The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
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17
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Son MY, Choi H, Han YM, Sook Cho Y. Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency. Stem Cells 2013; 31:2374-87. [DOI: 10.1002/stem.1509] [Citation(s) in RCA: 93] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2013] [Revised: 07/11/2013] [Accepted: 07/15/2013] [Indexed: 12/12/2022]
Affiliation(s)
- Mi-Young Son
- Stem Cell Research Center; Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon Republic of Korea
- Department of Biological Sciences; Korea Advanced Institute of Science and Technology (KAIST); Daejeon Republic of Korea
| | - Hoonsung Choi
- Stem Cell Research Center; Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon Republic of Korea
| | - Yong-Mahn Han
- Department of Biological Sciences; Korea Advanced Institute of Science and Technology (KAIST); Daejeon Republic of Korea
| | - Yee Sook Cho
- Stem Cell Research Center; Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon Republic of Korea
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18
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DUXO, a novel double homeobox transcription factor, is a regulator of the gastrula organizer in human embryonic stem cells. Stem Cell Res 2012; 9:261-9. [PMID: 23010573 DOI: 10.1016/j.scr.2012.08.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2012] [Revised: 08/03/2012] [Accepted: 08/09/2012] [Indexed: 01/04/2023] Open
Abstract
Human embryonic stem cells differentiate into gastrula organizer cells that express typical markers and induce secondary axes when injected into frog embryos. Here, we report that these human organizer cells express DUXO (DUX of the Organizer), a novel member of the double-homeobox (DUX) family of transcription factors, a group of genes unique to placental mammals. Both of DUXO's homeodomains share high similarity with those of Siamois and Twin, the initial inducers of the amphibian gastrula organizer. DUXO overexpression in human embryoid bodies induces organizer related genes, whereas its knock down hampers formation of the organizer and its derivatives. Finally, we show that DUXO regulates GOOSECOID, the canonical organizer marker, in a direct manner, suggesting that DUXO is a major regulator of human organizer formation.
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19
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Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL. Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues. PLoS One 2012; 7:e41564. [PMID: 22855692 PMCID: PMC3404047 DOI: 10.1371/journal.pone.0041564] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2012] [Accepted: 06/27/2012] [Indexed: 12/05/2022] Open
Abstract
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.
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Affiliation(s)
- Lili C. Kudo
- NeuroInDx, Inc., Signal Hill, California, United States of America
- * E-mail: (LCK); (SLK)
| | - Nancy Vi
- NeuroInDx, Inc., Signal Hill, California, United States of America
| | - Zhongcai Ma
- NeuroInDx, Inc., Signal Hill, California, United States of America
- Division of Neuroscience, Department of Neurology, Los Angeles Biomedical Research Institute at Harbor-University of California Los Angeles (UCLA) Medical Center, Torrance, California, United States of America
| | - Tony Fields
- Department of Neurology, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California, United States of America
| | - Nuraly K. Avliyakulov
- Department of Biological Chemistry, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California, United States of America
| | - Michael J. Haykinson
- Department of Biological Chemistry, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California, United States of America
| | - Anatol Bragin
- NeuroInDx, Inc., Signal Hill, California, United States of America
- Department of Neurology, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California, United States of America
| | - Stanislav L. Karsten
- NeuroInDx, Inc., Signal Hill, California, United States of America
- * E-mail: (LCK); (SLK)
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20
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Sharivkin R, Walker MD, Soen Y. Proteomics-based dissection of human endoderm progenitors by differential cell capture on antibody array. Mol Cell Proteomics 2012; 11:586-95. [PMID: 22580589 DOI: 10.1074/mcp.m111.016840] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomics platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated differential cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. We used this platform to fractionate the hitherto unresolved early endoderm compartment of CXCR4+ cells and identify several endoderm (CD61+ and CD63+) and non-endoderm (CD271+, CD49F+, CD44+ and B2M+) sub-populations. We provide evidence that one of these sub-populations, CD61+, is directly derived from CXCR4+ cells, displays characteristic kinetics of emergence, and exhibits a distinct gene expression profile. The results demonstrate the potential of the cell-capture antibody array as a powerful proteomics tool for detailed dissection of heterogeneous cellular systems.
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Affiliation(s)
- Revital Sharivkin
- Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel
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21
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Kopper O, Benvenisty N. Stepwise differentiation of human embryonic stem cells into early endoderm derivatives and their molecular characterization. Stem Cell Res 2012; 8:335-45. [DOI: 10.1016/j.scr.2011.12.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/18/2011] [Revised: 12/08/2011] [Accepted: 12/12/2011] [Indexed: 01/07/2023] Open
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22
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Modeling neurological disorders by human induced pluripotent stem cells. J Biomed Biotechnol 2011; 2011:350131. [PMID: 22162635 PMCID: PMC3227533 DOI: 10.1155/2011/350131] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2011] [Accepted: 10/06/2011] [Indexed: 01/30/2023] Open
Abstract
Studies of human brain development are critical as research on neurological disorders have been progressively advanced. However, understanding the process of neurogenesis through analysis of the early embryo is complicated and limited by a number of factors, including the complexity of the embryos, availability, and ethical constrains. The emerging of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) has shed light of a new approach to study both early development and disease pathology. The cells behave as precursors of all embryonic lineages; thus, they allow tracing the history from the root to individual branches of the cell lineage tree. Systems for neural differentiation of hESCs and iPSCs have provided an experimental model that can be used to augment in vitro studies of in vivo brain development. Interestingly, iPSCs derived from patients, containing donor genetic background, have offered a breakthrough approach to study human genetics of neurodegenerative diseases. This paper summarizes the recent reports of the development of iPSCs from patients who suffer from neurological diseases and evaluates the feasibility of iPSCs as a disease model. The benefits and obstacles of iPSC technology are highlighted in order to raising the cautions of misinterpretation prior to further clinical translations.
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23
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Toh YC, Blagovic K, Yu H, Voldman J. Spatially organized in vitro models instruct asymmetric stem cell differentiation. Integr Biol (Camb) 2011; 3:1179-87. [PMID: 22028041 DOI: 10.1039/c1ib00113b] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Understanding developmental biology requires knowledge of both the environmental factors regulating stem cell differentiation, which are increasingly being defined, and their spatial organization within a structurally heterogeneous niche, which is still largely unknown. Here we introduce spatially organized stem cell developmental models to interrogate the role of space in fate specification. Specifically, we developed Differential Environmental Spatial Patterning (δESP) to organize different microenvironments around single embryonic stem cell (ESC) colonies via sequential micropatterning. We first used δESP to decouple and understand the roles of cell organization and niche organization on ESCs deciding between self-renewal and differentiation fate choices. We then approximated in vitro an embryonic developmental step, specifically proximal-distal (PD) patterning of the mouse epiblast at pre-gastrulation, by spatially organizing two extraembryonic environments around ESCs, demonstrating that spatial organization of these three cell types is sufficient for PD patterns to form in vitro.
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Affiliation(s)
- Yi-Chin Toh
- Research Laboratory of Electronics, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA
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24
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Sharon N, Mor I, Golan-lev T, Fainsod A, Benvenisty N. Molecular and Functional Characterizations of Gastrula Organizer Cells Derived from Human Embryonic Stem Cells. Stem Cells 2011; 29:600-8. [DOI: 10.1002/stem.621] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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