Serum-Free Differentiation of Murine Embryonic Stem Cells into Alveolar Type II Epithelial Cells

Abstract

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.

NKX2-1 Activation by SMAD2 Signaling After Definitive Endoderm Differentiation in Human Embryonic Stem Cell

“…NKX2-1 can be induced in mouse (m) or human embryonic stem cell (hESC) in vitro [10][11][12][13][14][15][16]. Some studies have suggested that Activin-A [12,13] positively influences the differentiation ability of m/hESC towards NKX2-1 expressing pulmonary epithelium, whereas other studies suggest that pulmonary epithelium specification from the ventral foregut requires dual inactivation of BMP4/transforming growth factor (TGF) beta.…”

“…Some studies have suggested that Activin-A [12,13] positively influences the differentiation ability of m/hESC towards NKX2-1 expressing pulmonary epithelium, whereas other studies suggest that pulmonary epithelium specification from the ventral foregut requires dual inactivation of BMP4/transforming growth factor (TGF) beta. Some but not all studies have demonstrated that high concentrations of FGF2 increase NKX2-1 expression in differentiating hESC [9,10,15].…”

Directed Differentiation of Mesendoderm Derivatives from Embryonic Stem Cells

“…However, the lung epithelial cells derived by this method had a gene expression profile resembling that of the lung committed precursor (SPC + Ttf1 + ) found in foregut endoderm, rather than mature alveolar epithelium. While trying to optimize serum free conditions for lung epithelial cell differentiation, Winkler et al (2008) observed in the presence of serum relatively high expression levels of surfactant proteins A, B, C and D, CCSP and aquaporin 5, suggesting that epithelial cells expressing the markers for mature type II alveolar epithelium cells could be generated in serum-containing conditions. Surprisingly, a late treatment with ActivinA following serum activation resulted in significantly higher expression levels of SPC compared to an early induction with ActivinA.…”

The Future of Cell Therapy and Tissue Engineering in Cardiovascular Disease: The New Era of Biological Therapeutics

“…It has been shown that four factors present in ESC are sufficient to re-induce a pluripotent state in somatic cells. The reprogramming of somatic cells to a pluripotent state can be achieved by simple retroviral overexpression of specific transcription factors, resulting in induced pluripotent stem (iPS) cells that are almost indistinguishable from ESC (Okita K 2007;Narazaki G 2008;Winkler ME 2008). Transduction of fibroblasts with only four transcription factors, Oct4, Sox2, cMyc and Klf4, could dedifferentiate the fibroblasts to cells with almost all features of ESC.…”