Browsing by Subject "Embryonic stem cells"

Now showing 1 - 8 of 8
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    CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoid
    (Springer Nature, 2022-11-17) Nie, Jing; Ueda, Yoshitomo; Solivais, Alexander J.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors. Further analysis of the mutant otic progenitors suggests that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells reveal disruption of deafness gene expression as a potential underlying mechanism of CHARGE-associated sensorineural hearing loss. Notably, co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescues mutant phenotypes by restoring otherwise severely dysregulated otic genes. Taken together, our results suggest that CHD7 plays a critical role in regulating human otic lineage specification and hair cell differentiation.
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    Differentiation of pluripotent stem cells into endothelial cells
    (Wolters Kluwer, 2015-05) Yoder, Mervin C.; Department of Pediatrics, IU School of Medicine
    PURPOSE OF REVIEW: Methods to isolate endothelial cells from murine and human pluripotent stem cells continue to evolve and increasingly diverse endothelial cell populations have been generated. This review provides an update of key articles published within the past year that report on some of those advances. RECENT FINDINGS: Cooperative interactions among microRNA (miRNA), transcription factors and some downstream interacting proteins have been reported to enhance endothelial specification from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Endothelial cell differentiation can also be modulated by various growth factor additions, Notch pathway activation or inhibition, and modulation of the microenvironment of the differentiating ESC and iPSC. Functionality of the derived endothelium has been demonstrated by a variety of in-vitro and in-vivo assays. Finally, two recent reports have identified endothelial progenitor populations with robust proliferative potential. SUMMARY: Progress in differentiating endothelial cells from ESC and iPSC has been made. The recent report of formation of endothelial colony forming cells from human ESC and iPSC provides a protocol that can generate clinically relevant numbers of cells for human cell therapy.
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    Diverse Perspectives: Considerations About Embryonic Stem Cell Research
    (2006-09-01T18:18:29Z) Indiana University Center for Bioethics, Stem Cell Study Group
    Since the initial isolation of human embryonic stem cells in 1998 (Thomson et al. 1998), important developments in research have offered the promise of valuable therapeutic breakthroughs while continuing to raise significant social, ethical, legal and policy challenges. Among the interests of the Indiana University Center for Bioethics (IUCB) is a desire to engage issues of this kind, and in so doing, to provide a resource to the IU community, to Indiana, and to the entire country. The topic of stem cell research was, therefore, an appropriate one for discussion at the Center. In January 2002, the IUCB created a Stem Cell Study Group (SCSG). Our primary goal was to provide a forum for informed public discussion of the issues by making use of the considerable local scientific, legal and ethical expertise. In other words, we wanted primarily to educate ourselves about these issues. Our secondary goal was to identify and describe those points on which agreement could be achieved, as well as those issues on which agreement proved difficult if not impossible. This paper summarizes our efforts to meet both of these goals.
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    Reconstitution of mouse inner ear sensory development from pluripotent stem cells
    (2014-01) Koehler, Karl R.; Oxford, Gerry S.; Cummins, Theodore R.; Hashino, Eri; Meyer, Jason S.; Zhang, Xin
    The inner ear contains specialized sensory epithelia that detect head movements, gravity and sound. Hearing loss and imbalance are primarily caused by degeneration of the mechanosensitive hair cells in sensory epithelia or the sensory neurons that connect the inner ear to the brain. The controlled derivation of inner ear sensory epithelia and neurons from pluripotent stem cells will be essential for generating in vitro models of inner ear disorders or developing cell-based therapies. Despite some recent success in deriving hair cells from mouse embryonic stem (ES) cells, it is currently unclear how to derive inner ear sensory cells in a fully defined and reproducible manner. Progress has likely been hindered by what is known about induction of the nonneural and preplacodal ectoderm, two critical precursors during inner ear development. The studies presented here report the step-wise differentiation of inner ear sensory epithelia from mouse ES cells in three-dimensional culture. We show that nonneural, preplacodal and pre-otic epithelia can be generated from ES cell aggregates by precise temporal control of BMP, TGFβ and FGF signaling, mimicking in vivo development. Later, in a self-guided process, vesicles containing supporting cells emerge from the presumptive otic epithelium and give rise to hair cells with stereocilia bundles and kinocilium. Remarkably, the vesicles developed into large cysts with sensory epithelia reminiscent of vestibular sense organs (i.e. the utricle, saccule and crista), which sense head movements and gravity in the animal. We have designated these stem cell-derived structures inner ear organoids. In addition, we discovered that sensory-like neurons develop alongside the organoids and form putative synapses with hair cells in a similar fashion to the hair cell-to-neuron circuit that forms in the developing embryo. Our data thus establish a novel in vitro model of inner ear organogenesis that can be used to gain deeper insight into inner ear development and disorder.
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    The role of CFP1 in murine embryonic stem cell function and liver regeneration
    (2015-08) Mahadevan, Jyothi; Skalnik, David G.; Goebl, Mark G.; Harrington, Maureen A.; Herring, B. Paul
    CXXC finger protein 1 (Cfp1), a component of the Set1 histone methyltransferase complex, is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryos lacking Cfp1 are unable to gastrulate and Cfp1-null embryonic stem (ES) cells fail to undergo cellular differentiation in vitro. However, expression of wild type Cfp1 in Cfp1-null ES cells rescues differentiation capacity, suggesting that dynamic epigenetic changes occurring during lineage specification require Cfp1. The domain structure of Cfp1 consists of a DNA binding CXXC domain and an N-terminal plant homeodomain (PHD). PHDs are frequently observed in chromatin remodeling proteins, functioning as reader modules for histone marks. However, the histone binding properties and underlying functional significance of Cfp1 PHD are largely unknown. My research revealed that Cfp1 PHD directly and specifically binds to histone H3K4me1/me2/me3 marks. A point mutation that abolishes binding to methylated H3K4 (W49A) does not affect rescue of cellular differentiation, but, point mutations that abolish both methylated H3K4 (W49A) and DNA (C169A) binding result in defective in vitro differentiation, indicating that PHD and CXXC exhibit redundant functions. The mammalian liver has the unique ability to regenerate following injury. Previous studies indicated that Cfp1 is essential for hematopoiesis in zebrafish and mice. I hypothesized that Cfp1 additionally plays a role in liver development and regeneration. To understand the importance of Cfp1 in liver development and regeneration, I generated a mouse line lacking Cfp1 specifically in the liver (Cfp1fl/fl Alb-Cre+). Around 40% of these mice display a wasting phenotype and die within a year. Livers of these mice have altered global H3K4me3 levels and often exhibit regenerative nodules. Most importantly, livers of these mice display an impaired regenerative response following partial hepatectomy. Collectively, these findings establish Cfp1 as an epigenetic regulator essential for ES cell function and liver homeostasis and regeneration.
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    SIRT1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress
    (Wiley, 2014-05) Ou, Xuan; Lee, Man Ryul; Huang, Xinxin; Messina-Graham, Steven; Broxmeyer, Hal E.; Department of Microbiology & Immunology, School of Medicine
    SIRT1, an NAD-dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age-related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2 O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-, than in WT mESCs. However, addition of 3-methyladenine, a widely used autophagy inhibitor, in combination with H2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II, lowered expression of Beclin-1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways.
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    Structure-function analysis of CXXC finger protein 1
    (2009-04) Tate, Courtney Marie; Skalnik, David Gordon; Bigsby, Robert M.; Dynlacht, Joseph R.; Wek, Ronald C.
    This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.
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    Transcriptional Regulation of Retinal Progenitor Cells Derived from Human Induced Pluripotent Stem Cells.
    (2013-08-22) Sridhar, Akshayalakshmi; Meyer, Jason S.; Marrs, James; Belecky-Adams, Teri
    In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.