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      Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein

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          Abstract

          Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that regulates chloride and water transport across all epithelia and affects multiple organs including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction-coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof-of-concept, treatment of CF patient induced pluripotent stem cells (iPSC)-derived epithelial cells with a novel small molecule compound to correct for the common CF-processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.

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          Most cited references28

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          Basal cells as stem cells of the mouse trachea and human airway epithelium.

          The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER(T2) transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.
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            Preparing for the first breath: genetic and cellular mechanisms in lung development.

            The mammalian respiratory system--the trachea and the lungs--arises from the anterior foregut through a sequence of morphogenetic events involving reciprocal endodermal-mesodermal interactions. The lung itself consists of two highly branched, tree-like systems--the airways and the vasculature--that develop in a coordinated way from the primary bud stage to the generation of millions of alveolar gas exchange units. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as branching morphogenesis, vascular development, and the differentiation of multipotent progenitor populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding respiratory disorders, congenital defects in human neonates, and how the disruption of morphogenetic programs early in lung development can lead to deficiencies that persist throughout life. (c) 2010 Elsevier Inc. All rights reserved.
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              Vertebrate endoderm development and organ formation.

              The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                3 March 2014
                September 2012
                21 April 2014
                : 30
                : 9
                : 876-882
                Affiliations
                [1 ]Program in Developmental & Stem Cell Biology, Hospital for Sick Children, Toronto, ON, M5G 1L7
                [2 ]Ontario Human Induced Pluripotent Stem Cell Facility, Toronto, ON, M5G 1L7
                [3 ]Program in Molecular Structure and Function, Hospital for Sick Children, Toronto
                [4 ]Program in Physiology & Experimental Medicine, Hospital for Sick Children, Toronto
                [5 ]Division of Respiratory Medicine, Hospital for Sick Children, Toronto
                [6 ]Department of Molecular Genetics, University of Toronto, Toronto, ON, M5S 1A8
                [7 ]Department of Obstetrics and Gynaecology, University of Toronto, Toronto
                Author notes
                Please address inquiries to: Janet Rossant, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8, Phone: 416-813-7929, Fax: 416-813-5085, janet.rossant@ 123456sickkids.ca Or James Ellis, Hospital for Sick Children, 101 College Street, TMDT @ MaRS 13-710, Toronto, Ontario, Canada M5G 1L7, Phone: 416-813-7295, Fax: 416-813-5252, jellis@ 123456sickkids.ca
                Article
                CAMS3958
                10.1038/nbt.2328
                3994104
                22922672
                94389aaa-e8c9-4328-9239-bf3d1708f44b
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                Biotechnology
                Biotechnology

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