Generation of lung epithelial-like tissue from human embryonic stem cells

Haute, Lindsey Van; Block, Gert De; Liebaers, Ingeborg; Sermon, Karen; Rycke, Martine De

doi:10.1186/1465-9921-10-105

Cited by 80 publications

(73 citation statements)

References 25 publications

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“…Such an ALI is commonly used to push the maturation of human primary bronchial epithelial cells in vitro and has been used to assist in the differentiation of embryonic stem cells to lung epithelial cells (2,35,36). The ALI was maintained in media G on the basolateral side (37) and used to promote maturation and polarization of the epithelial cell layer.…”

Section: Resultsmentioning

confidence: 99%

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Firth

Dargitz

Qualls

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

221

214

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Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium. iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC. The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro. Grant Funding Source: Supported in part by CIRM and the Berger Foundation

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Section: Resultsmentioning

confidence: 99%

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Firth

Dargitz

Qualls

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

221

214

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“…Thus, transfection and culture procedures have been developed which facilitate the differentiation of hESCs into a pure (> 99%) population of ATII cells with morphological characteristics including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the CF transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5 (Wang et al, 2007). Protocols have also been developed whereby human embryonic stem cells can be differentiated into the major cell types of lung epithelial tissue (Van Haute et al, 2009). ATII cells have also been derived from iPSCs and have phenotypic properties similar to mature human alveolar type II cells (iPS-ATII) (Ghaedi et al, 2014).…”

Section: Stem Cell-derived Lung Cellsmentioning

confidence: 99%

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

Gordon

Daneshian

Bouwstra

et al. 2015

ALTEX

124

103

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SummaryModels of the outer epithelia of the human body -namely the skin, the intestine and the lung -have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.

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“…We used TaqMan assays from Applied Biosystems to detect RNA levels for the following genes: CXADR (Hs00154661_m1); SOX2 (Hs01053049_s1); NANOG (Hs02387400_g1); COLL1 (Hs00164004_m1); RUNX2 (Hs00231692_m1); GAPDH (Hs99999905_m1) and RPS24 (Hs03006009_g1). Primers and probes for UBC (F: 5 0 -CGCAGCCGGGATTTG-3 0 ; R: 5 0 -TCAAGTGACGATCA-CAGCGA-3 0 ; probe TCGCAGTTCTTGTTTGTG) and POU5-F1_iA (F: 5 0 -GGACACCTGGTCTGCGATTT-3 0 ; R: 5 0 -CAT-CACCTCCACCACCTGG-3 0 ; probe GCCTTCTCGCCCCC) were self-designed (Van Haute et al 2009), while those for KRT7 (F:…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions

Krivega¹,

Geens²,

Velde³

2014

Reproduction

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Coxsackie virus and adenovirus receptor, CXADR (CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.

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Generation of lung epithelial-like tissue from human embryonic stem cells

Cited by 80 publications

References 25 publications

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions

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