Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.
Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.
Winkler
Monica E
ME
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany.
Mauritz
Christina
C
Groos
Stephanie
S
Kispert
Andreas
A
Menke
Sandra
S
Hoffmann
Anika
A
Gruh
Ina
I
Schwanke
Kristin
K
Haverich
Axel
A
Martin
Ulrich
U
D000465:Algorithms; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D037701:Pulmonary Surfactant-Associated Protein B / Q000378:metabolism; D037721:Pulmonary Surfactant-Associated Protein C / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000502:physiology; D044967:Serum / Q000502:physiology; D013997:Time Factors
D016895:Culture Media, Serum-Free; D037701:Pulmonary Surfactant-Associated Protein B; D037721:Pulmonary Surfactant-Associated Protein C