Generation of lung epithelial-like tissue from human embryonic stem cells.
BACKGROUND
Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.


METHODS
Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.


RESULTS
Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.


CONCLUSIONS
These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.
Van Haute
Lindsey
L
Department of Embryology and Genetics, UZ Brussel, Belgium. lindsey.vanhaute@uzbrussel.be
De Block
Gert
G
Liebaers
Inge
I
Sermon
Karen
K
De Rycke
Martine
M
D051402:Aquaporin 5 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008168:Lung / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism; D037721:Pulmonary Surfactant-Associated Protein C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000074482:Thyroid Nuclear Factor 1; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D014598:Uteroglobin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
C496036:AQP5 protein, human; D051402:Aquaporin 5; D015415:Biomarkers; C106349:FOXJ1 protein, human; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; D037662:Pulmonary Surfactant-Associated Protein A; D037721:Pulmonary Surfactant-Associated Protein C; D012333:RNA, Messenger; C556389:SCGB1A1 protein, human; C489679:SFTPC protein, human; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D014404:Tubulin; D014746:Vimentin; D014598:Uteroglobin