Generation of Functional Neutrophils from a Mouse Model of X-Linked Chronic Granulomatous Disorder Using Induced Pluripotent Stem Cells
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{"title"=>"Generation of functional neutrophils from a mouse model of X-linked chronic granulomatous disorder using induced pluripotent stem cells", "type"=>"journal", "authors"=>[{"first_name"=>"Sayandip", "last_name"=>"Mukherjee", "scopus_author_id"=>"7401816522"}, {"first_name"=>"Giorgia", "last_name"=>"Santilli", "scopus_author_id"=>"55907715700"}, {"first_name"=>"Michael P.", "last_name"=>"Blundell", "scopus_author_id"=>"7004126464"}, {"first_name"=>"Susana", "last_name"=>"Navarro", "scopus_author_id"=>"14322422600"}, {"first_name"=>"Juan A.", "last_name"=>"Bueren", "scopus_author_id"=>"7003849270"}, {"first_name"=>"Adrian J.", "last_name"=>"Thrasher", "scopus_author_id"=>"7005085037"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-79952299565", "doi"=>"10.1371/journal.pone.0017565", "sgr"=>"79952299565", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"21408614", "issn"=>"19326203", "pui"=>"361382335"}, "id"=>"925b1bc6-be3a-34be-bb2c-90278d99dd98", "abstract"=>"Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.", "link"=>"http://www.mendeley.com/research/generation-functional-neutrophils-mouse-model-xlinked-chronic-granulomatous-disorder-using-induced-p-4", "reader_count"=>24, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>1, "Researcher"=>10, "Student > Ph. D. Student"=>7, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>1, "Researcher"=>10, "Student > Ph. D. Student"=>7, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>19, "Medicine and Dentistry"=>1, "Neuroscience"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>19}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Iran"=>1, "Brazil"=>1, "Italy"=>1, "India"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/794038"], "description"=>"<p>(A) Schematic representation of protocol for haematopoietic differentiation of X-CGD mouse derived iPS cells. EB, embryoid bodies; ColIV, collagen type IV coated plate; Flk-1+ cells, FMS-like tyrosine kinase I positive cells; OP9, M-CSF deficient OP9 stromal cell line; LSK, Lineage negative, Sca-1 positive, c-kit positive cells. (B) Characterization of day six EB cultured under low-attachment conditions. Semi-quantitative RT-PCR analyses showing comparative expression of pluripotency and differentiation markers in wild-type (wt-IPSC) and diseased iPS cells (cgd-IPSC). Left panel shows expression levels of various markers in day six EBs, while those in the right panel shows comparative levels of expression in cells after culturing in collagen IV coated flasks in haematopoietic differentiation medium. Middle panel shows in-situ immunophenotypic staining of six-day EBs with antibodies targeting characteristic antigens expressed in the three germinal layers. (C) FACS analysis showing generation of Flk-1 positive cells from EBs upon collagen IV culture.(D) FACS plot showing derivation of lineage negative, Sca-1 positive and c-kit positive (LSK) population post OP9 co-culture of Flk-1 positive cells.</p>", "links"=>[], "tags"=>["differentiation", "ips", "cells", "mesodermal", "haematopoietic"], "article_id"=>464400, "categories"=>["Immunology"], "users"=>["Sayandip Mukherjee", "Giorgia Santilli", "Michael P. Blundell", "Susana Navarro", "Juan A. Bueren", "Adrian J. Thrasher"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0017565.g002", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vitro_differentiation_of_iPS_cells_towards_mesodermal_and_haematopoietic_lineages_/464400", "title"=>"In-vitro differentiation of iPS cells towards mesodermal and haematopoietic lineages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 20:09:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/794142"], "description"=>"<p>Colony Forming Unit (CFU) assay was performed to test the ability of LSK cells to undergo terminal differentiation in semi-solid methylcellulose media containing suitable cytokine cocktail. (A) Graph showing the relative distribution of CFU-GM (granulocyte/macrophage), CFU-G (granulocyte), and CFU-M (macrophage) colonies scored on the basis of their morphology from three independent cultures. Error bars denote standard error of mean (SEM). (B) Representative cytospin preparation of Diff-Quik stained cells isolated from characteristic colonies showing presence of granulocytes and macrophages including and not restricted to mature and functional neutrophils. (C) FACS plot showing presence of double positive Gr-1 (granulocyte differentiation marker) and CD11b (myeloid and NK cells marker) cells isolated from colonies growing in methycellulose differentiation medium.</p>", "links"=>[], "tags"=>["differentiation", "ips", "derived", "lsk"], "article_id"=>464513, "categories"=>["Immunology"], "users"=>["Sayandip Mukherjee", "Giorgia Santilli", "Michael P. Blundell", "Susana Navarro", "Juan A. Bueren", "Adrian J. Thrasher"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0017565.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myeloid_differentiation_of_iPS_cell_derived_LSK_cells_/464513", "title"=>"Myeloid differentiation of iPS cell derived LSK cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 20:10:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/794242"], "description"=>"<p>(A) Phase contrast images showing results of Nitroblue tetrazolium (NBT) assay. Left panel showing untransduced NBT negative (i) and LentiSFFVgp91 transduced NBT positive (ii) colony. Graph showing positive correlation between the multiplicity of infection (m.o.i) and generation of NBT positive colonies. (B) Comparative analysis of the efficiency of disease phenotype correction by lentiviral transduction of pre- and post-reprogramming targets (fibroblasts vs EBs respectively), as assayed by percentage of NBT positive colonies. Restoration of NADPH oxidase activity was compared to number of NBT positive colonies obtained from wild-type iPSC derived myeloid progenitors. Error bars denote standard error of mean (SEM). (C) Agarose gel electrophoresis image showing PCR amplification of SFFV promoter sequence in genomic DNA of NBT negative CFUs confirming the presence of integrated vector. UT, untransduced; LV, Lentivector transduced. (D) Bisulphite genomic sequencing analysis showing higher degree of methylation in lentiviral promoter (SFFV) when vector transduction was performed at the fibroblast stage compared to EB stage. Filled boxes indicate methylated CpG dinucleotide.</p>", "links"=>[], "tags"=>["x-cgd", "phenotype"], "article_id"=>464610, "categories"=>["Immunology"], "users"=>["Sayandip Mukherjee", "Giorgia Santilli", "Michael P. Blundell", "Susana Navarro", "Juan A. Bueren", "Adrian J. Thrasher"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0017565.g004", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Correction_of_X_CGD_phenotype_in_vitro_/464610", "title"=>"Correction of X-CGD phenotype in-vitro.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 20:11:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/793919"], "description"=>"<p>(A) Images of iPSC showing ES cell like morphology (high nucleus to cytoplasm ratio), high levels of alkaline phosphatase (AP) activity, and expression of pluripotency markers Sox2, Oct4, Klf4, Nanog, SSEA-1, and c-Myc. Bright-field images were acquired with a standard Olympus microscope (20X objective). Fluorescent images were acquired with a Zeiss LSM 710 confocal microscope (25X objective). (B) Haematoxylin & Eosin staining of teratoma sections showing derivatives from three germinal layers (Ecto, ectoderm; Endo, endoderm; Meso, mesoderm). Structures shown include neural tube (ectodermal), striated muscles and cartilaginous structures (mesodermal), and gut-like epithelium (endodermal). (C) Immunofluorescent confocal images of teratoma sections showing antibody staining targeting tissue derivatives present in three germinal layers.Tuj1: neuronal class III β-tubulin; ∝-FP: alpha-fetoprotein; ∝-act: alpha-actinin; DAPI: 4′-6-Diamidino-2-phenylindole.(D) Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses showing silencing of exogenously introduced transgenes as shown by their presence in fibroblasts (three days post transduction, D3), and absence in cgd-IPSC clone (passage five). Specific primers were designed to target regions of retroviral transgenes and endogenous sequences of reprogramming factors. GAPDH, glyceraldehdye 3-phosphate dehydrogenase.</p>", "links"=>[], "tags"=>["x-cgd", "fibroblasts", "induced", "pluripotent"], "article_id"=>464277, "categories"=>["Immunology"], "users"=>["Sayandip Mukherjee", "Giorgia Santilli", "Michael P. Blundell", "Susana Navarro", "Juan A. Bueren", "Adrian J. Thrasher"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0017565.g001", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reprogramming_of_X_CGD_mouse_fibroblasts_to_induced_pluripotent_stem_cells_/464277", "title"=>"Reprogramming of X-CGD mouse fibroblasts to induced pluripotent stem cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 20:09:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/794334"], "description"=>"<p>Embryoid bodies were transduced with lentiviral vector encoding codon optimized <i>gp91<sup>phox</sup></i> from an internal chimeric promoter (LentiChimgp91). (A) Graph showing positive correlation between the multiplicity of infection (m.o.i) and generation of NBT positive colonies. Error bars denote standard error of mean (SEM). Phase contrast images of CFU-GM colonies showing results from NBT assay. (B) FACS derived histograms showing results of dihydrorhodamine123 (DHR) test upon phorbol myristate acetate (PMA) stimulation. DHR loaded neutrophils upon PMA stimulation show a shift in fluorescence due to reduction of DHR123 by respiratory burst generated by functional neutrophils.</p>", "links"=>[], "tags"=>["lentiviral", "vector", "disease-free", "haematopoietic"], "article_id"=>464701, "categories"=>["Immunology"], "users"=>["Sayandip Mukherjee", "Giorgia Santilli", "Michael P. Blundell", "Susana Navarro", "Juan A. Bueren", "Adrian J. Thrasher"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0017565.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficacy_of_novel_lentiviral_vector_for_generation_of_disease_free_haematopoietic_cells_/464701", "title"=>"Efficacy of novel lentiviral vector for generation of disease-free haematopoietic cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 20:11:41"}

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Relative Metric

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