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1
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Singh A, Rappolee DA, Ruden DM. Epigenetic Reprogramming in Mice and Humans: From Fertilization to Primordial Germ Cell Development. Cells 2023; 12:1874. [PMID: 37508536 PMCID: PMC10377882 DOI: 10.3390/cells12141874] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Revised: 07/10/2023] [Accepted: 07/11/2023] [Indexed: 07/30/2023] Open
Abstract
In this review, advances in the understanding of epigenetic reprogramming from fertilization to the development of primordial germline cells in a mouse and human embryo are discussed. To gain insights into the molecular underpinnings of various diseases, it is essential to comprehend the intricate interplay between genetic, epigenetic, and environmental factors during cellular reprogramming and embryonic differentiation. An increasing range of diseases, including cancer and developmental disorders, have been linked to alterations in DNA methylation and histone modifications. Global epigenetic reprogramming occurs in mammals at two stages: post-fertilization and during the development of primordial germ cells (PGC). Epigenetic reprogramming after fertilization involves rapid demethylation of the paternal genome mediated through active and passive DNA demethylation, and gradual demethylation in the maternal genome through passive DNA demethylation. The de novo DNA methyltransferase enzymes, Dnmt3a and Dnmt3b, restore DNA methylation beginning from the blastocyst stage until the formation of the gastrula, and DNA maintenance methyltransferase, Dnmt1, maintains methylation in the somatic cells. The PGC undergo a second round of global demethylation after allocation during the formative pluripotent stage before gastrulation, where the imprints and the methylation marks on the transposable elements known as retrotransposons, including long interspersed nuclear elements (LINE-1) and intracisternal A-particle (IAP) elements are demethylated as well. Finally, DNA methylation is restored in the PGC at the implantation stage including sex-specific imprints corresponding to the sex of the embryo. This review introduces a novel perspective by uncovering how toxicants and stress stimuli impact the critical period of allocation during formative pluripotency, potentially influencing both the quantity and quality of PGCs. Furthermore, the comprehensive comparison of epigenetic events between mice and humans breaks new ground, empowering researchers to make informed decisions regarding the suitability of mouse models for their experiments.
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Affiliation(s)
- Aditi Singh
- CS Mott Center, Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48202, USA; (A.S.); (D.A.R.)
- Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48202, USA
| | - Daniel A. Rappolee
- CS Mott Center, Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48202, USA; (A.S.); (D.A.R.)
- Reproductive Stress Measurement, Mechanisms and Management, Corp., 135 Lake Shore Rd., Grosse Pointe Farms, MI 48236, USA
- Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48202, USA
- Department of Physiology, Wayne State University, Detroit, MI 48202, USA
| | - Douglas M. Ruden
- CS Mott Center, Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48202, USA; (A.S.); (D.A.R.)
- Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48202, USA
- Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48202, USA
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2
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Oh JN, Jeong J, Lee M, Choe GC, Lee DK, Choi KH, Kim SH, Lee CK. Characterization of multitype colonies originating from porcine blastocysts produced in vitro. Front Cell Dev Biol 2022; 10:918222. [PMID: 36172290 PMCID: PMC9510650 DOI: 10.3389/fcell.2022.918222] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Accepted: 08/01/2022] [Indexed: 11/30/2022] Open
Abstract
Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.
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Affiliation(s)
- Jong-Nam Oh
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Jinsol Jeong
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Mingyun Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Gyung Cheol Choe
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Dong-Kyung Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Kwang-Hwan Choi
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Seung-Hun Kim
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Chang-Kyu Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
- Designed Animal and Transplantation Research Institute (DATRI), Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang, South Korea
- *Correspondence: Chang-Kyu Lee,
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3
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Kumar D, Talluri TR, Selokar NL, Hyder I, Kues WA. Perspectives of pluripotent stem cells in livestock. World J Stem Cells 2021; 13:1-29. [PMID: 33584977 PMCID: PMC7859985 DOI: 10.4252/wjsc.v13.i1.1] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/28/2020] [Accepted: 11/09/2020] [Indexed: 02/06/2023] Open
Abstract
The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.
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Affiliation(s)
- Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India.
| | - Thirumala R Talluri
- Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner 334001, India
| | - Naresh L Selokar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India
| | - Iqbal Hyder
- Department of Physiology, NTR College of Veterinary Science, Gannavaram 521102, India
| | - Wilfried A Kues
- Department of Biotechnology, Friedrich-Loeffler-Institute, Federal Institute of Animal Health, Neustadt 31535, Germany
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4
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Sozen B, Demir N, Zernicka-Goetz M. BMP signalling is required for extra-embryonic ectoderm development during pre-to-post-implantation transition of the mouse embryo. Dev Biol 2020; 470:84-94. [PMID: 33217407 DOI: 10.1016/j.ydbio.2020.11.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Revised: 11/06/2020] [Accepted: 11/08/2020] [Indexed: 12/12/2022]
Abstract
At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis.
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Affiliation(s)
- Berna Sozen
- Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK; California Institute of Technology, Division of Biology and Biological Engineering, 1200 E. California Boulevard, Pasadena, CA, 91125, USA; Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, 07070, Turkey; Yale University School of Medicine, Department of Genetics, New Haven, CT, 06510, USA
| | - Necdet Demir
- Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, 07070, Turkey
| | - Magdalena Zernicka-Goetz
- Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK; California Institute of Technology, Division of Biology and Biological Engineering, 1200 E. California Boulevard, Pasadena, CA, 91125, USA.
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Roberts RM, Ezashi T, Sheridan MA, Yang Y. Specification of trophoblast from embryonic stem cells exposed to BMP4. Biol Reprod 2019; 99:212-224. [PMID: 29579154 DOI: 10.1093/biolre/ioy070] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Accepted: 03/21/2018] [Indexed: 01/16/2023] Open
Abstract
Trophoblast (TB) comprises the outer cell layers of the mammalian placenta that make direct contact with the maternal uterus and, in species with a highly invasive placenta, maternal blood. It has its origin as trophectoderm, a single epithelial layer of extra-embryonic ectoderm that surrounds the embryo proper at the blastocyst stage of development. Here, we briefly compare the features of TB specification and determination in the mouse and the human. We then review research on a model system that has been increasingly employed to study TB emergence, namely the BMP4 (bone morphogenetic protein-4)-directed differentiation of human embryonic stem cells (ESCd), and discuss why outcomes using it have proved so uneven. We also examine the controversial aspects of this model, particularly the issue of whether or not the ESCd represents TB at all. Our focus here has been to explore similarities and potential differences between the phenotypes of ESCd, trophectoderm, placental villous TB, and human TB stem cells. We then explore the role of BMP4 in the differentiation of human pluripotent cells to TB and suggest that it converts the ESC into a totipotent state that is primed for TB differentiation when self-renewal is blocked. Finally we speculate that the TB formed from ESC is homologous to the trophectoderm-derived, invasive TB that envelopes the implanting conceptus during the second week of pregnancy.
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Affiliation(s)
- R Michael Roberts
- Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.,Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.,Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Toshihiko Ezashi
- Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.,Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Megan A Sheridan
- Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.,Department of Biochemistry, University of Missouri, Columbia, Missouri, USA
| | - Ying Yang
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, USA
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6
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Nandi P, Lim H, Torres-Garcia EJ, Lala PK. Human trophoblast stem cell self-renewal and differentiation: Role of decorin. Sci Rep 2018; 8:8977. [PMID: 29895842 PMCID: PMC5997742 DOI: 10.1038/s41598-018-27119-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 05/24/2018] [Indexed: 12/18/2022] Open
Abstract
The origin and regulation of stem cells sustaining trophoblast renewal in the human placenta remain unclear. Decorin, a leucine-rich proteoglycan restrains trophoblast proliferation, migration/invasiveness and endovascular differentiation, and local decorin overproduction is associated with preeclampsia (PE). Here, we tested the role of decorin in human trophoblast stem cell self-renewal and differentiation, using two models: an immortalized first trimester trophoblast cell line HTR-8/SVneo (HTR) and freshly isolated primary trophoblast (p-trophoblast) from early first trimester (6-9 weeks) placentas. Self-renewal capacity was measured by spheroid forming ability of single cells on ultra-low attachment plates for multiple generations. Markers of embryonic stem (ES) cells, trophoblast stem (TS) cells and trophoblast were used to identify stem cell hierarchy. Differentiation markers for syncytial and extravillous (EVT) pathways were employed to identify differentiated cells. Bewo cells were additionally used to explore DCN effects on syncytialization. Results reveal that the incidence of spheroid forming stem-like cells was 13-15% in HTR and 0.1-0.4%, in early first trimester p-trophoblast, including a stem cell hierarchy of two populations of ES and TS-like cells. DCN restrained ES cell self-renewal, promoted ES to TS transition and maintenance of TS cell stem-ness, but inhibited TS cell differentiation into both syncytial and EVT pathways.
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Affiliation(s)
- Pinki Nandi
- Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
| | - Hyobin Lim
- Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
| | - Eloy Jose Torres-Garcia
- Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
| | - Peeyush K Lala
- Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
- Department of Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
- Associate Scientist, Children's Health Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
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7
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Cloutier M, Harris C, Gayen S, Maclary E, Kalantry S. Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation. Methods Mol Biol 2018; 1861:177-203. [PMID: 30218368 PMCID: PMC6209079 DOI: 10.1007/978-1-4939-8766-5_14] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.
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Affiliation(s)
- Marissa Cloutier
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Clair Harris
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Srimonta Gayen
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Emily Maclary
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
- Department of Biology, University of Utah, Salt Lake City, UT, USA
| | - Sundeep Kalantry
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA.
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8
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Roberts RM, Yuan Y, Ezashi T. Exploring early differentiation and pluripotency in domestic animals. Reprod Fertil Dev 2017; 29:101-107. [PMID: 28278797 DOI: 10.1071/rd16292] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
This short review describes some general features of the origins of the pluripotent inner cell mass and epiblast during the early development of eutherian mammals and the two kinds of embryonic stem cell (ESC), naïve and primed type, that have been produced from these structures. We point out that the derivation of pluripotent stem cells from domesticated species continues to be fraught with difficulties, most likely because the culture requirements of these cells are distinct from those of mouse and human ESCs. Generation of induced pluripotent stem cells (iPSCs) from the domesticated species has been more straightforward, although the majority of the iPSC lines remain dependent on the continued expression of one or more integrated reprogramming genes. Although hope for the potential usefulness of these cells in genetic modification of livestock and other domestic species has dimmed, ESCs and iPSCs remain our best source of self-renewing populations of pluripotent cells, with potential usefulness in preserving and propagating valuable animal breeds and making contributions to fields such as regenerative medicine, toxicology and even laboratory meat production.
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Affiliation(s)
- R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, 245 Bond Life Sciences Center, 1201 East Rollins Street, Columbia, MO 65211, USA
| | - Ye Yuan
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, 245 Bond Life Sciences Center, 1201 East Rollins Street, Columbia, MO 65211, USA
| | - Toshihiko Ezashi
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, 245 Bond Life Sciences Center, 1201 East Rollins Street, Columbia, MO 65211, USA
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9
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Chang CW, Parast MM. Human trophoblast stem cells: Real or not real? Placenta 2017; 60 Suppl 1:S57-S60. [PMID: 28087122 DOI: 10.1016/j.placenta.2017.01.003] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/24/2016] [Revised: 12/30/2016] [Accepted: 01/04/2017] [Indexed: 10/24/2022]
Abstract
Abnormal trophoblast differentiation is the root cause of many placenta-based pregnancy complications, including preeclampsia and fetal growth restriction. Human trophoblast differentiation is difficult to study due to the lack of a stem cell model. Such a multipotent "trophoblast stem" (TS) cell, with the ability to differentiate into all trophoblast subtypes, has been derived from mouse blastocysts, but attempts to derive similar human cells have failed. We consider here several possibilities for the TS cell niche in the human placenta. Aside from discussion of such a niche in the pre-implantation blastocyst, we discuss evidence for these TS cells residing in the post-implantation villous cytotrophoblast layer, or even in the non-trophoblast portions, of the human placenta. It is our hope that recognition of the niche would lead to successful derivation and in vitro establishment of such cells, which could then be disseminated widely to the placental biology community for advancing the field. Availability of self-renewing human TS cells, whose gene expression and environment could be manipulated, will provide a platform, not just for the study of pathophysiology of placental disease, but also for the discovery of diagnostic biomarkers and therapeutic targets for common pregnancy complications.
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Affiliation(s)
- Ching-Wen Chang
- Department of Pathology, University of California San Diego, La Jolla, CA, USA; Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA, USA
| | - Mana M Parast
- Department of Pathology, University of California San Diego, La Jolla, CA, USA; Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA, USA.
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10
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Motomura K, Inoue K, Ogura A. Selection of accurate reference genes in mouse trophoblast stem cells for reverse transcription-quantitative polymerase chain reaction. J Reprod Dev 2016; 62:311-5. [PMID: 26853688 PMCID: PMC4919296 DOI: 10.1262/jrd.2015-170] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Mouse trophoblast stem cells (TSCs) form colonies of different sizes and morphologies, which might reflect
their degrees of differentiation. Therefore, each colony type can have a characteristic gene expression
profile; however, the expression levels of internal reference genes may also change, causing fluctuations in
their estimated gene expression levels. In this study, we validated seven housekeeping genes by using a
geometric averaging method and identified Gapdh as the most stable gene across different
colony types. Indeed, when Gapdh was used as the reference, expression levels of
Elf5, a TSC marker gene, stringently classified TSC colonies into two groups: a high
expression groups consisting of type 1 and 2 colonies, and a lower expression group consisting of type 3 and 4
colonies. This clustering was consistent with our putative classification of undifferentiated/differentiated
colonies based on their time-dependent colony transitions. By contrast, use of an unstable reference gene
(Rn18s) allowed no such clear classification. Cdx2, another TSC marker,
did not show any significant colony type-specific expression pattern irrespective of the reference gene.
Selection of stable reference genes for quantitative gene expression analysis might be critical, especially
when cell lines consisting of heterogeneous cell populations are used.
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Affiliation(s)
- Kaori Motomura
- RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
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11
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Oliveira F, Sangalli J, Meirelles F, Perecin F, Silva Filho P, Watanabe Y, Miglino M, Assis Neto A. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage. ARQ BRAS MED VET ZOO 2015. [DOI: 10.1590/1678-4162-7647] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.
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12
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Abstract
This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably stem from an inability to maintain the stability of the endogenous gene networks necessary to maintain pluripotency. Based on work in humans and rodents, achievement of full pluripotency will likely require fine adjustments in the growth factors and signaling inhibitors provided to the cells. Finally, we discuss the future utility of these cells for biomedical and agricultural purposes.
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Affiliation(s)
- Toshihiko Ezashi
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - Ye Yuan
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
| | - R Michael Roberts
- Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; , ,
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13
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14
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Li Y, Parast MM. BMP4 regulation of human trophoblast development. THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 2015; 58:239-46. [PMID: 25023690 DOI: 10.1387/ijdb.130341mp] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Since the derivation of human embryonic stem cells, and the subsequent generation of induced pluripotent stem cells, there has been much excitement about the ability to model and evaluate human organ development in vitro. The finding that these cells, when treated with BMP4, are able to generate the extraembryonic cell type, trophoblast, which is the predominant functional epithelium in the placenta, has not been widely accepted. This review evaluates this model, providing comparison to early known events during placentation in both human and mouse and addresses specific challenges. Keeping in mind the ultimate goal of understanding human placental development and pregnancy disorders, our aim here is two-fold: to distinguish gaps in our knowledge arising from mis- or over-interpretation of data, and to recognize the limitations of both mouse and human models, but to work within those limitations towards the ultimate goal.
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Affiliation(s)
- Yingchun Li
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
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15
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Wu T, Liu Y, Wen D, Tseng Z, Tahmasian M, Zhong M, Rafii S, Stadtfeld M, Hochedlinger K, Xiao A. Histone variant H2A.X deposition pattern serves as a functional epigenetic mark for distinguishing the developmental potentials of iPSCs. Cell Stem Cell 2014; 15:281-294. [PMID: 25192463 DOI: 10.1016/j.stem.2014.06.004] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 04/27/2014] [Accepted: 06/05/2014] [Indexed: 01/05/2023]
Abstract
For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines.
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Affiliation(s)
- Tao Wu
- Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA
| | - Yifei Liu
- Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA
| | - Duancheng Wen
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY 10065, USA
| | - Zito Tseng
- Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA
| | - Martik Tahmasian
- Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA
| | - Mei Zhong
- Yale Stem Cell Center and Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520, USA
| | - Shahin Rafii
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY 10065, USA
| | - Matthias Stadtfeld
- Massachusetts General Hospital Cancer Center, Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Medicine, Harvard University, Boston, MA 02114, USA
| | - Konrad Hochedlinger
- Massachusetts General Hospital Cancer Center, Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Medicine, Harvard University, Boston, MA 02114, USA
| | - Andrew Xiao
- Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.
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16
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Petkova R, Arabadjiev B, Chakarov S, Pankov R. Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse? BIOTECHNOL BIOTEC EQ 2014; 28:184-191. [PMID: 26019504 PMCID: PMC4434091 DOI: 10.1080/13102818.2014.907037] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2013] [Accepted: 11/14/2013] [Indexed: 01/15/2023] Open
Abstract
The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells.
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Affiliation(s)
- Rumena Petkova
- Scientific Technological Service (STS) Ltd., Sofia, Bulgaria
| | - Borislav Arabadjiev
- Scientific Technological Service (STS) Ltd., Sofia, Bulgaria
- Department of Cell Biology, Histology and Embryology, and Department of Biochemistry, Faculty of Biology, Sofia University ‘St. Kliment Ohridsky’, Sofia, Bulgaria
| | - Stoyan Chakarov
- Department of Cell Biology, Histology and Embryology, and Department of Biochemistry, Faculty of Biology, Sofia University ‘St. Kliment Ohridsky’, Sofia, Bulgaria
| | - Roumen Pankov
- Department of Cell Biology, Histology and Embryology, and Department of Biochemistry, Faculty of Biology, Sofia University ‘St. Kliment Ohridsky’, Sofia, Bulgaria
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17
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Senner CE, Krueger F, Oxley D, Andrews S, Hemberger M. DNA methylation profiles define stem cell identity and reveal a tight embryonic-extraembryonic lineage boundary. Stem Cells 2013; 30:2732-45. [PMID: 23034951 DOI: 10.1002/stem.1249] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2012] [Revised: 08/28/2012] [Accepted: 09/08/2012] [Indexed: 01/27/2023]
Abstract
Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.
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Affiliation(s)
- Claire E Senner
- Epigenetics Programme, The Babraham Institute, Cambridge, UK
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18
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Li Y, Moretto-Zita M, Soncin F, Wakeland A, Wolfe L, Leon-Garcia S, Pandian R, Pizzo D, Cui L, Nazor K, Loring JF, Crum CP, Laurent LC, Parast MM. BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state. Development 2013; 140:3965-76. [PMID: 24004950 DOI: 10.1242/dev.092155] [Citation(s) in RCA: 102] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63(+)/KRT7(+) CTB stem cell state, followed by formation of functional KLF4(+) STB and HLA-G(+) EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63(+) CTB stem cells to terminally differentiated trophoblast subtypes.
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Affiliation(s)
- Yingchun Li
- Department of Pathology, University of California San Diego, La Jolla, CA 92093, USA
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19
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Trophoblast lineage cells derived from human induced pluripotent stem cells. Biochem Biophys Res Commun 2013; 436:677-84. [PMID: 23774580 DOI: 10.1016/j.bbrc.2013.06.016] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2013] [Accepted: 06/06/2013] [Indexed: 11/23/2022]
Abstract
BACKGROUND During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient's placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. METHODS AND RESULTS Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. CONCLUSION Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.
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20
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Sehgal P, Chaturvedi P, Kumaran RI, Kumar S, Parnaik VK. Lamin A/C haploinsufficiency modulates the differentiation potential of mouse embryonic stem cells. PLoS One 2013; 8:e57891. [PMID: 23451281 PMCID: PMC3581495 DOI: 10.1371/journal.pone.0057891] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2012] [Accepted: 01/28/2013] [Indexed: 02/06/2023] Open
Abstract
Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development.
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Affiliation(s)
- Poonam Sehgal
- CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
| | | | - R. Ileng Kumaran
- CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
| | - Satish Kumar
- CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
| | - Veena K. Parnaik
- CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
- * E-mail:
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21
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Moerkamp AT, Paca A, Goumans MJ, Kunath T, Kruithof BPT, Kruithof-de Julio M. Extraembryonic endoderm cells as a model of endoderm development. Dev Growth Differ 2013; 55:301-8. [PMID: 23414197 DOI: 10.1111/dgd.12036] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2012] [Revised: 12/19/2012] [Accepted: 12/19/2012] [Indexed: 02/06/2023]
Abstract
In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm-like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.
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Affiliation(s)
- Asja T Moerkamp
- Department of Molecular and Cell Biology, Centre of Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands
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22
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Wang Q, Cooney AJ. The Role of Nuclear Receptors in Embryonic Stem Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 786:287-306. [DOI: 10.1007/978-94-007-6621-1_16] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
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23
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Epigenetic regulation of stem cells : the role of chromatin in cell differentiation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 786:307-28. [PMID: 23696364 DOI: 10.1007/978-94-007-6621-1_17] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The specialized cell types of tissues and organs are generated during development and are replenished over lifetime though the process of differentiation. During differentiation the characteristics and identity of cells are changed to meet their functional requirements. Differentiated cells then faithfully maintain their characteristic gene expression patterns. On the molecular level transcription factors have a key role in instructing specific gene expression programs. They act together with chromatin regulators which stabilize expression patterns. Current evidence indicates that epigenetic mechanisms are essential for maintaining stable cell identities. Conversely, the disruption of chromatin regulators is associated with disease and cellular transformation. In mammals, a large number of chromatin regulators have been identified. The Polycomb group complexes and the DNA methylation system have been widely studied in development. Other chromatin regulators remain to be explored. This chapter focuses on recent advances in understanding epigenetic regulation in embryonic and adult stem cells in mammals. The available data illustrate that several chromatin regulators control key lineage specific genes. Different epigenetic systems potentially could provide stability and guard against loss or mutation of individual components. Recent experiments also suggest intervals in cell differentiation and development when new epigenetic patterns are established. Epigenetic patterns have been observed to change at a progenitor state after stem cells commit to differentiation. This finding is consistent with a role of epigenetic regulation in stabilizing expression patterns after their establishment by transcription factors. However, the available data also suggest that additional, presently unidentified, chromatin regulatory mechanisms exist. Identification of these mechanism is an important aim for future research to obtain a more complete framework for understanding stem cell differentiation during tissue homeostasis.
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24
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Liu Y, Timani K, Ou X, Broxmeyer HE, He JJ. C-MYC controlled TIP110 protein expression regulates OCT4 mRNA splicing in human embryonic stem cells. Stem Cells Dev 2012; 22:689-94. [PMID: 23088399 DOI: 10.1089/scd.2012.0271] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.
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Affiliation(s)
- Ying Liu
- Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, Texas 76092, USA.
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25
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Harel S, Tu EY, Weisberg S, Esquilin M, Chambers SM, Liu B, Carson CT, Studer L, Reizis B, Tomishima MJ. ZFX controls the self-renewal of human embryonic stem cells. PLoS One 2012; 7:e42302. [PMID: 22879936 PMCID: PMC3411758 DOI: 10.1371/journal.pone.0042302] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2011] [Accepted: 07/06/2012] [Indexed: 11/18/2022] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.
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Affiliation(s)
- Sivan Harel
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America
| | - Edmund Y. Tu
- Developmental Biology Program, Sloan-Kettering Institute, New York, New York, United States of America
| | - Stuart Weisberg
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America
| | - Manuel Esquilin
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America
| | - Stuart M. Chambers
- Developmental Biology Program, Sloan-Kettering Institute, New York, New York, United States of America
| | - Becky Liu
- Developmental Biology Program, Sloan-Kettering Institute, New York, New York, United States of America
| | | | - Lorenz Studer
- Developmental Biology Program, Sloan-Kettering Institute, New York, New York, United States of America
| | - Boris Reizis
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America
- * E-mail: (BR); (MJT)
| | - Mark J. Tomishima
- Developmental Biology Program, Sloan-Kettering Institute, New York, New York, United States of America
- * E-mail: (BR); (MJT)
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26
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Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells. In Vitro Cell Dev Biol Anim 2012; 48:403-6. [DOI: 10.1007/s11626-012-9525-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2012] [Accepted: 06/13/2012] [Indexed: 10/28/2022]
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27
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Leeb M, Wutz A. Establishment of epigenetic patterns in development. Chromosoma 2012; 121:251-62. [PMID: 22427185 PMCID: PMC3350763 DOI: 10.1007/s00412-012-0365-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2011] [Revised: 02/24/2012] [Accepted: 02/24/2012] [Indexed: 12/14/2022]
Abstract
The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic "memory" of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation.
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Affiliation(s)
- Martin Leeb
- Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
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28
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Kuckenberg P, Kubaczka C, Schorle H. The role of transcription factor Tcfap2c/TFAP2C in trophectoderm development. Reprod Biomed Online 2012; 25:12-20. [PMID: 22560121 DOI: 10.1016/j.rbmo.2012.02.015] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2011] [Revised: 01/23/2012] [Accepted: 02/22/2012] [Indexed: 11/17/2022]
Abstract
In recent years, knowledge regarding the genetic and epigenetic programmes governing specification, maintenance and differentiation of the extraembryonic lineage has advanced substantially. Establishment and analysis of mice deficient in genes implicated in trophoblast lineage and the option to generate and manipulate murine stem cell lines from the inner cell mass and the trophectoderm in vitro represent major advances. The activating enhancer binding protein 2 (AP2) family of transcription factors is expressed during mammalian development and in certain malignant diseases. This article summarizes the data regarding expression and function of murine Tcfap2 and human TFAP2 in extraembryonic development and differentiation. It also presents a model integrating Tcfap2c into the framework of trophoblast development and highlights the requirement of Tcfap2c to maintain trophoblast stem cells. With regard to human trophoblast cell-lineage restriction, the role of TFAP2C in lineage specification and maintenance is speculated upon. Furthermore, an overview of target genes of AP2 in mouse and human affecting placenta development and function is provided and the evidence suggesting that defects in regulating TFAP2 members might contribute to placental defects is discussed.
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Affiliation(s)
- Peter Kuckenberg
- Institute of Pathology, Department of Developmental Pathology, University of Bonn, Germany
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29
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Stem cells giving rise to extraembryonic tissues. Bull Exp Biol Med 2012; 150:504-14. [PMID: 22268054 DOI: 10.1007/s10517-011-1179-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
The review is devoted to characterization of stem cells involved in the formation of extraembryonic tissues during the early development of mammalian embryos. Here we present our results of characterization of stem cells from the trophoblast and extraembryonic endoderm of voles and comparative analysis of these cells and the corresponding mouse cells and discuss possible signal pathways maintaining these cells in undifferentiated state.
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30
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Tachibana M, Sparman M, Ramsey C, Ma H, Lee HS, Penedo MCT, Mitalipov S. Generation of chimeric rhesus monkeys. Cell 2012; 148:285-95. [PMID: 22225614 DOI: 10.1016/j.cell.2011.12.007] [Citation(s) in RCA: 126] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2011] [Revised: 10/25/2011] [Accepted: 12/05/2011] [Indexed: 01/12/2023]
Abstract
Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.
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Affiliation(s)
- Masahito Tachibana
- Oregon National Primate Research Center, Oregon Health & Science University, 505 N.W. 185(th) Avenue, Beaverton, OR 97006, USA
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31
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Martinez Arias A, Brickman JM. Gene expression heterogeneities in embryonic stem cell populations: origin and function. Curr Opin Cell Biol 2011; 23:650-6. [PMID: 21982544 DOI: 10.1016/j.ceb.2011.09.007] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2011] [Revised: 09/08/2011] [Accepted: 09/13/2011] [Indexed: 11/15/2022]
Abstract
Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal.
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32
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Jordan NV, Johnson GL, Abell AN. Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer. Cell Cycle 2011; 10:2865-73. [PMID: 21862874 DOI: 10.4161/cc.10.17.17188] [Citation(s) in RCA: 180] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Epithelial-mesenchymal transition (EMT) is an essential developmental program that becomes reactivated in adult tissues to promote the progression of cancer. EMT has been largely studied by examining the beginning epithelial state or the ending mesenchymal state without studying the intermediate stages. Recent studies using trophoblast stem (TS) cells paused in EMT have defined the molecular and epigenetic mechanisms responsible for modulating the intermediate "metastable" stages of EMT. Targeted inactivation of MAP3K4, knockdown of CBP, or overexpression of SNAI1 in TS cells induced similar metastable phenotypes. These TS cells exhibited epigenetic changes in the histone acetylation landscape that cause loss of epithelial maintenance while preserving self-renewal and multipotency. A similar phenotype was found in claudin-low breast cancer cells with properties of EMT and stemness. This intersection between EMT and stemness in TS cells and claudin-low metastatic breast cancer demonstrates the usefulness of developmental EMT systems to understand EMT in cancer.
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Affiliation(s)
- Nicole Vincent Jordan
- Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
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Macaulay EC, Weeks RJ, Andrews S, Morison IM. Hypomethylation of functional retrotransposon-derived genes in the human placenta. Mamm Genome 2011; 22:722-35. [PMID: 21874386 DOI: 10.1007/s00335-011-9355-1] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Accepted: 07/29/2011] [Indexed: 11/25/2022]
Abstract
DNA hypomethylation is assumed to be a feature of the mammalian placenta; however, its role in regulating placental gene expression is not well defined. In this study, MeDIP and Sequenom MassARRAY were used to identify hypomethylated gene promoters in the human placenta. Among the genes identified, the hypomethylation of an alternative promoter for KCNH5 was found to be restricted to the placenta and chorion. Complete methylation of this promoter correlates with a silenced KCNH5 transcript in embryonic tissues, including the amnion. Unusually, this hypomethylated promoter and the alternative first exon are derived from a SINE (AluY) retrotransposon. Examination of additional retrotransposon-derived gene promoters in the placenta confirmed that retrotransposon hypomethylation permits the placenta-specific expression of these genes. Furthermore, the lineage-specific methylation displayed by KCNH5, INSL4, and ERVWE1 revealed that dichotomous methylation establishes differential retrotransposon silencing between the extra-embryonic and embryonic lineages. The hypomethylation of the retrotransposons that regulate these genes, each of which arose during recent primate evolution, is consistent with these genes having functional roles that are unique to the invasive haemochorial placentas of humans and recent primates.
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Affiliation(s)
- Erin C Macaulay
- Department of Pathology, Dunedin School of Medicine and National Research Centre for Growth and Development, University of Otago, New Zealand
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XU HEN, ADDIS RUSSELLC, GOINGS DAVIDAF, NIOKA SHOKO, CHANCE BRITTON, GEARHART JOHND, LI LINZ. IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES. JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES 2011; 4:279-288. [PMID: 34046096 PMCID: PMC8153411 DOI: 10.1142/s1793545811001617] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/15/2023]
Abstract
Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ∼200-440 μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.
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Affiliation(s)
- HE N. XU
- Molecular Imaging Laboratory, Department of
Radiology University of Pennsylvania, School of Medicine Philadelphia, PA 19104,
USA
- Britton Chance Laboratory of Redox Imaging Johnson
Research Foundation Department of Biochemistry and Biophysics University of
Pennsylvania, School of Medicine Philadelphia, PA 19104, USA
| | - RUSSELL C. ADDIS
- Institute for Regenerative Medicine and Department
of Cell and Developmental Biology University of Pennsylvania, School of Medicine
Philadelphia, PA 19104, USA
| | - DAVIDA F. GOINGS
- Institute for Regenerative Medicine and Department
of Cell and Developmental Biology University of Pennsylvania, School of Medicine
Philadelphia, PA 19104, USA
| | - SHOKO NIOKA
- Britton Chance Laboratory of Redox Imaging Johnson
Research Foundation Department of Biochemistry and Biophysics University of
Pennsylvania, School of Medicine Philadelphia, PA 19104, USA
| | - BRITTON CHANCE
- Britton Chance Laboratory of Redox Imaging Johnson
Research Foundation Department of Biochemistry and Biophysics University of
Pennsylvania, School of Medicine Philadelphia, PA 19104, USA
| | - JOHN D. GEARHART
- Institute for Regenerative Medicine and Department
of Cell and Developmental Biology University of Pennsylvania, School of Medicine
Philadelphia, PA 19104, USA
| | - LIN Z. LI
- Molecular Imaging Laboratory, Department of
Radiology University of Pennsylvania, School of Medicine Philadelphia, PA 19104,
USA
- Institute for Regenerative Medicine and Department
of Cell and Developmental Biology University of Pennsylvania, School of Medicine
Philadelphia, PA 19104, USA
- Britton Chance Laboratory of Redox Imaging Johnson
Research Foundation Department of Biochemistry and Biophysics University of
Pennsylvania, School of Medicine Philadelphia, PA 19104, USA
- Institute of Translational Medicine and Therapeutics
University of Pennsylvania, Philadelphia, PA 19104, USA
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Li X, Zhu L, Yang A, Lin J, Tang F, Jin S, Wei Z, Li J, Jin Y. Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell 2011; 8:46-58. [PMID: 21211781 DOI: 10.1016/j.stem.2010.11.027] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2010] [Revised: 10/02/2010] [Accepted: 10/25/2010] [Indexed: 11/18/2022]
Abstract
Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.
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Affiliation(s)
- Xiang Li
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine, Shanghai, 200025, China
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Andrecut M, Halley JD, Winkler DA, Huang S. A general model for binary cell fate decision gene circuits with degeneracy: indeterminacy and switch behavior in the absence of cooperativity. PLoS One 2011; 6:e19358. [PMID: 21625586 PMCID: PMC3098230 DOI: 10.1371/journal.pone.0019358] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Accepted: 03/31/2011] [Indexed: 11/30/2022] Open
Abstract
Background The gene regulatory circuit motif in which two opposing fate-determining transcription factors inhibit each other but activate themselves has been used in mathematical models of binary cell fate decisions in multipotent stem or progenitor cells. This simple circuit can generate multistability and explains the symmetric “poised” precursor state in which both factors are present in the cell at equal amounts as well as the resolution of this indeterminate state as the cell commits to either cell fate characterized by an asymmetric expression pattern of the two factors. This establishes the two alternative stable attractors that represent the two fate options. It has been debated whether cooperativity of molecular interactions is necessary to produce such multistability. Principal Findings Here we take a general modeling approach and argue that this question is not relevant. We show that non-linearity can arise in two distinct models in which no explicit interaction between the two factors is assumed and that distinct chemical reaction kinetic formalisms can lead to the same (generic) dynamical system form. Moreover, we describe a novel type of bifurcation that produces a degenerate steady state that can explain the metastable state of indeterminacy prior to cell fate decision-making and is consistent with biological observations. Conclusion The general model presented here thus offers a novel principle for linking regulatory circuits with the state of indeterminacy characteristic of multipotent (stem) cells.
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Affiliation(s)
- Mircea Andrecut
- Institute for Biocomplexity and Informatics, University of Calgary, Calgary, Alberta, Canada
| | - Julianne D. Halley
- Institute for Biocomplexity and Informatics, University of Calgary, Calgary, Alberta, Canada
| | - David A. Winkler
- Commonwealth Scientific and Industrial Research Organisation (CSIRO) Materials Science and Engineering, Clayton, Australia
- Monash Institute for Pharmaceutical Science, Parkville, Australia
- * E-mail: (SH); (DAW)
| | - Sui Huang
- Institute for Biocomplexity and Informatics, University of Calgary, Calgary, Alberta, Canada
- * E-mail: (SH); (DAW)
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37
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38
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Erb TM, Schneider C, Mucko SE, Sanfilippo JS, Lowry NC, Desai MN, Mangoubi RS, Leuba SH, Sammak PJ. Paracrine and epigenetic control of trophectoderm differentiation from human embryonic stem cells: the role of bone morphogenic protein 4 and histone deacetylases. Stem Cells Dev 2011; 20:1601-14. [PMID: 21204619 DOI: 10.1089/scd.2010.0281] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Our understanding of paracrine and epigenetic control of trophectoderm (TE) differentiation is limited by available models of preimplantation human development. Simple, defined media for selective TE differentiation of human embryonic stem cells (hESCs) were developed, enabling mechanistic studies of early placental development. Paracrine requirements of preimplantation human development were evaluated with hESCs by measuring lineage-specific transcription factor expression levels in single cells and morphological transformation in response to selected paracrine and epigenetic modulators. Bone morphogenic protein 4 (BMP4) addition to feeder-free pluripotent stem cells on matrigel frequently formed CDX2-positive TE. However, BMP4 or activin A inhibition alone also produced a mix of mesoderm and extraembryonic endoderm under these conditions. Further, BMP4 failed to form TE from adherent hESC maintained in standard feeder-dependent monolayers. Given that the efficiency and selectivity of BMP4-induced TE depended on medium components, we developed a basal medium containing insulin and heparin. In this medium, BMP4 induction of TE was dose dependent and with activin A inhibition by SB431542 (SB), approached 100% of cells. This paracrine stimulation of pluripotent cells transformed colony morphology from a cuboidal to squamous epithelium quantitatively on day 3, and produced significant multinucleated syncytiotrophoblasts by day 8. Addition of trichostatin A, a histone deacetylase (HDAC) inhibitor, reduced HDAC3, histone H3K9 methylation, and slowed differentiation in a dose-dependent manner. Modulators of BMP4- or HDAC-dependent signaling might adversely influence the timing and viability of early blastocyst developed in vitro. Since blastocyst development is synchronized to uterine receptivity, epigenetic regulators of TE differentiation might adversely affect implantation in vivo.
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Affiliation(s)
- Teresa M Erb
- Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee Women's Hospital of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
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40
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von Melchner H, Stewart AF. Methods for extracting function from mammalian genomes. Methods 2011; 53:329-30. [PMID: 21392581 DOI: 10.1016/j.ymeth.2011.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/01/2011] [Indexed: 10/18/2022] Open
Affiliation(s)
- Harald von Melchner
- Department for Molecular Hematology, University of Frankfurt Medical School, 60590 Frankfurt am Main, Germany.
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41
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Garcia-Tuñon I, Guallar D, Alonso-Martin S, Benito AA, Benítez-Lázaro A, Pérez-Palacios R, Muniesa P, Climent M, Sánchez M, Vidal M, Schoorlemmer J. Association of Rex-1 to target genes supports its interaction with Polycomb function. Stem Cell Res 2011; 7:1-16. [PMID: 21530438 DOI: 10.1016/j.scr.2011.02.005] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2010] [Revised: 02/21/2011] [Accepted: 02/23/2011] [Indexed: 12/31/2022] Open
Abstract
Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.
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Affiliation(s)
- I Garcia-Tuñon
- Regenerative Medicine Programme, IIS Aragón, Instituto Aragonés de Ciencias de la Salud, Zaragoza, Avda. Gómez Laguna, 25, Pl. 11, 50009 Zaragoza, Spain
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42
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Addis RC, Prasad MK, Yochem RL, Zhan X, Sheets TP, Axelman J, Patterson ES, Shamblott MJ. OCT3/4 regulates transcription of histone deacetylase 4 (Hdac4) in mouse embryonic stem cells. J Cell Biochem 2011; 111:391-401. [PMID: 20506506 DOI: 10.1002/jcb.22707] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4+ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells.
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Affiliation(s)
- Russell C Addis
- Department of Cell and Developmental Biology, and Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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43
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Renaud SJ, Karim Rumi MA, Soares MJ. Review: Genetic manipulation of the rodent placenta. Placenta 2011; 32 Suppl 2:S130-5. [PMID: 21256588 DOI: 10.1016/j.placenta.2010.12.017] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2010] [Revised: 12/10/2010] [Accepted: 12/13/2010] [Indexed: 10/18/2022]
Abstract
The principal role of the placenta is the maintenance of pregnancy and promotion of fetal growth and viability. The use of transgenic rodents has greatly enhanced our understanding of placental development and function. However, embryonic lethality is often a confounding variable in determining whether a genetic modification adversely affected placental development. In these cases, it is beneficial to specifically manipulate the placental genome. The purpose of this review is to summarize available methodologies for specific genetic modification of the rodent placenta. By restricting genetic alterations to the trophoblast lineage, it is possible to gain a deeper understanding of placental development that perhaps will lead to gene-targeted therapies to rescue irregular placentation in transgenic animals or in women at high-risk for placenta-associated pregnancy complications.
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Affiliation(s)
- S J Renaud
- Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA.
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44
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Papaioannou VE, Behringer RR. Early embryonic lethality in genetically engineered mice: diagnosis and phenotypic analysis. Vet Pathol 2011; 49:64-70. [PMID: 21233329 DOI: 10.1177/0300985810395725] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Embryonic lethality is a common phenotype that occurs in mice that are homozygous for genetically engineered mutations. These phenotypes highlight the time and place that a gene is first required during embryogenesis. Early embryonic lethality (ie, before and up to mid-gestation) can be straightforward to analyze because the stage at which death occurs suggests why an embryo has failed. Here we summarize general strategies for analyzing early embryonic lethal phenotypes in genetically engineered mouse mutants.
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Affiliation(s)
- V E Papaioannou
- Department of Genetics and Development, Columbia University Medical Center, 701 W 168th St, New York, NY 10032, USA.
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45
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Lu R, Yang A, Jin Y. Dual functions of T-box 3 (Tbx3) in the control of self-renewal and extraembryonic endoderm differentiation in mouse embryonic stem cells. J Biol Chem 2010; 286:8425-8436. [PMID: 21189255 DOI: 10.1074/jbc.m110.202150] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Embryonic stem cells (ESCs) possess the capacity to proliferate indefinitely in an undifferentiated state and to differentiate into various cell types in an organism. However, the critical question of how self-renewal and differentiation are precisely regulated in ESCs is not entirely understood at present. Here, we report the essential role of Tbx3, a pluripotency-related transcription factor of the T-box gene family, for both the maintenance of self-renewal of mouse ESCs and for their differentiation into extraembryonic endoderm (ExEn). We show that Tbx3 is highly expressed in ExEn cells in addition to undifferentiated ESCs. Knockdown of Tbx3 expression using tetracycline-regulated Tbx3 siRNA resulted in the attenuation of ESC self-renewal ability and aberrant differentiation processes, including reduced ExEn differentiation but enhanced ectoderm and trophectoderm differentiation. Conversely, inducible forced expression of Tbx3 triggered ExEn lineage commitment. Mechanistically, Tbx3 directly activated the expression of Gata6, an essential regulator of ExEn. Interestingly, Tbx3 modulated H3K27me3 modification and the association of the PRC2 complex with the promoter region of Gata6. Taken together, the results of this study revealed a previously unappreciated role of a pluripotency factor in ExEn differentiation. Additionally, our data reveal that Tbx3 may function through direct binding and epigenetic modification of histones on the Gata6 promoter to maintain the ExEn differentiation potential of ESCs.
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Affiliation(s)
- Rui Lu
- From the Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, CAS/Shanghai Jiao Tong University School of Medicine and
| | - Acong Yang
- From the Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, CAS/Shanghai Jiao Tong University School of Medicine and; the Shanghai Stem Cell Institute, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Ying Jin
- From the Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, CAS/Shanghai Jiao Tong University School of Medicine and; the Shanghai Stem Cell Institute, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
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46
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Understanding gene circuits at cell-fate branch points for rational cell reprogramming. Trends Genet 2010; 27:55-62. [PMID: 21146896 DOI: 10.1016/j.tig.2010.11.002] [Citation(s) in RCA: 151] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2010] [Revised: 11/03/2010] [Accepted: 11/03/2010] [Indexed: 11/21/2022]
Abstract
Cell-type reprogramming, the artificial induction of a switch of cell lineage and developmental stage, holds great promise for regenerative medicine. However, how does the metazoan body itself 'program' the various cell lineages in the first place? Knowledge of how multipotent cells make cell-fate decisions and commit to a particular lineage is crucial for a rational reprogramming strategy and to avoid trial-and-error approaches in choosing the appropriate set of transcription factors to use. In the past few years, a general principle has emerged in which small gene circuits of cross-inhibition and self-activation govern the decision at branch points of cell development. A formal theoretical treatment of such circuits that deal with their dynamics on the 'epigenetic landscape' could offer some guidance to find the optimal way of cell reprogramming.
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Abstract
Trophoblast stem cells (TSC) are the precursors of the differentiated cells of the placenta. In the mouse, TSC can be derived from outgrowths of either blastocyst polar trophectoderm (TE) or extraembryonic ectoderm (ExE), which originates from polar TE after implantation. The mouse TSC niche appears to be located within the ExE adjacent to the epiblast, on which it depends for essential growth factors, but whether this cellular architecture is the same in other species remains to be determined. Mouse TSC self-renewal can be sustained by culture on mitotically inactivated feeder cells, which provide one or more factors related to the NODAL pathway, and a medium supplemented with FGF4, heparin, and fetal bovine serum. Repression of the gene network that maintains pluripotency and emergence of the transcription factor pathways that specify a trophoblast (TR) fate enables TSC derivation in vitro and placental formation in vivo. Disrupting the pluripotent network of embryonic stem cells (ESC) causes them to default to a TR ground state. Pluripotent cells that have acquired sublethal chromosomal alterations may be sequestered into TR for similar reasons. The transition from ESC to TSC, which appears to be unidirectional, reveals important aspects of initial fate decisions in mice. TSC have yet to be derived from domestic species in which remarkable TR growth precedes embryogenesis. Recent derivation of TSC from blastocysts of the rhesus monkey suggests that isolation of the human equivalents may be possible and will reveal the extent to which mechanisms uncovered by using animal models are true in our own species.
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Affiliation(s)
- R Michael Roberts
- Division of Animal Sciences, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
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48
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Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui JI, Knisely AS, Hirabayashi M, Nakauchi H. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 2010; 142:787-99. [PMID: 20813264 DOI: 10.1016/j.cell.2010.07.039] [Citation(s) in RCA: 422] [Impact Index Per Article: 28.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2010] [Revised: 05/05/2010] [Accepted: 06/30/2010] [Indexed: 12/26/2022]
Abstract
The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.
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Affiliation(s)
- Toshihiro Kobayashi
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
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49
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No shortcuts to pig embryonic stem cells. Theriogenology 2010; 74:544-50. [DOI: 10.1016/j.theriogenology.2010.04.020] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2010] [Revised: 04/16/2010] [Accepted: 04/18/2010] [Indexed: 12/15/2022]
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50
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Bianco C, Rangel MC, Castro NP, Nagaoka T, Rollman K, Gonzales M, Salomon DS. Role of Cripto-1 in stem cell maintenance and malignant progression. THE AMERICAN JOURNAL OF PATHOLOGY 2010; 177:532-40. [PMID: 20616345 DOI: 10.2353/ajpath.2010.100102] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Cripto-1 is critical for early embryonic development and, together with its ligand Nodal, has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Like other embryonic genes, Cripto-1 performs important roles in the formation and progression of several types of human tumors, stimulating cell proliferation, migration, epithelial to mesenchymal transition, and tumor angiogenesis. Several studies have demonstrated that cell fate regulation during embryonic development and cell transformation during oncogenesis share common signaling pathways, suggesting that uncontrolled activation of embryonic signaling pathways might drive cell transformation and tumor progression in adult tissues. Here we review our current understanding of how Cripto-1 controls stem cell biology and how it integrates with other major embryonic signaling pathways. Because many cancers are thought to derive from a subpopulation of cancer stem-like cells, which may re-express embryonic genes, Cripto-1 signaling may drive tumor growth through the generation or expansion of tumor initiating cells bearing stem-like characteristics. Therefore, the Cripto-1/Nodal signaling may represent an attractive target for treatment in cancer, leading to the elimination of undifferentiated stem-like tumor initiating cells.
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Affiliation(s)
- Caterina Bianco
- Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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