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      In vitro generation of human pluripotent stem cell derived lung organoids


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          Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.

          DOI: http://dx.doi.org/10.7554/eLife.05098.001

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          Cell behavior has traditionally been studied in the lab in two-dimensional situations, where cells are grown in thin layers on cell-culture dishes. However, most cells in the body exist in a three-dimensional environment as part of complex tissues and organs, and so researchers have been attempting to re-create these environments in the lab. To date, several such ‘organoids’ have been successfully generated, including models of the human intestine, stomach, brain and liver. These organoids can mimic the responses of real tissues and can be used to investigate how organs form, change with disease, and how they might respond to potential therapies.

          Here, Dye et al. developed a new three-dimensional model of the human lung by coaxing human stem cells to become specific types of cells that then formed complex tissues in a petri dish. To make these lung organoids, Dye et al. manipulated several of the signaling pathways that control the formation of organs during the development of animal embryos. First, the stem cells were instructed to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and other several other internal organs.

          Then, Dye et al. activated two important developmental pathways that are known to make endoderm form three-dimensional intestinal tissue. However, by inhibiting two other key developmental pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos instead.

          This early lung-like tissue formed three-dimensional spherical structures as it developed. The next challenge was to make these structures develop into lung tissue. Dye et al. worked out a method to do this, which involved exposing the cells to additional proteins that are involved in lung development. The resulting lung organoids survived in laboratory cultures for over 100 days and developed into well-organized structures that contain many of the types of cells found in the lung.

          Further analysis revealed the gene activity in the lung organoids resembles that of the lung of a developing human fetus, suggesting that lung organoids grown in the dish are not fully mature. Dye et al.'s findings provide a new approach for creating human lung organoids in culture that may open up new avenues for investigating lung development and diseases.

          DOI: http://dx.doi.org/10.7554/eLife.05098.002

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          Most cited references67

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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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            What is principal component analysis?

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              Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.

              Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human beta-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with approximately 3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional beta-cells, including expression of critical beta-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell-derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.

                Author and article information

                Role: Reviewing editor
                eLife Sciences Publications, Ltd
                24 March 2015
                : 4
                [1 ]deptDepartment of Cell and Developmental Biology , University of Michigan Medical School , Ann Arbor, United States
                [2 ]deptDepartment of Internal Medicine , University of Michigan Medical School , Ann Arbor, United States
                [3 ]deptDivision of Developmental Biology , Cincinnati Children's Hospital Medical Center , Cincinnati, United States
                [4 ]deptDivision of Endocrinology , Cincinnati Children's Hospital Medical Center , Cincinnati, United States
                [5 ]deptInstitute for Human Genetics, Department of Pediatrics , University of California, San Francisco , San Francisco, United States
                [6 ]deptProgram in Craniofacial and Mesenchymal Biology , University of California, San Francisco , San Francisco, United States
                [7 ]deptCenter for Craniofacial Anomalies , University of California, San Francisco , San Francisco, United States
                [8 ]deptDepartment of Laboratories , Seattle Children's Hospital and University of Washington , Seattle, United States
                [9 ]deptCenter for Organogenesis , University of Michigan Medical School , Ann Arbor, United States
                University of Toronto , Canada
                University of Toronto , Canada
                Author notes
                [* ]For correspondence: spencejr@ 123456umich.edu
                © 2015, Dye et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Funded by: FundRef http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute (NHBLI);
                Award ID: R21HL115372
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000912, March of Dimes Foundation;
                Award ID: Basil O'Connor starter scholar award
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK);
                Award ID: K01DK091415
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute (NHBLI);
                Award ID: R01HL119215
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Research Article
                Developmental Biology and Stem Cells
                Custom metadata
                Directed differentiation of stem cells can generate ventral-anterior foregut spheroids that can expand into three-dimensional lung organoids with striking structural, cellular and molecular similarities to the human fetal lung.

                Life sciences
                pluripotent stem cells,organoids,endoderm,lung,foregut,spheroid,human
                Life sciences
                pluripotent stem cells, organoids, endoderm, lung, foregut, spheroid, human


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