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At this point, about 30% of cells expressed the cystic fibrosis transmembrane conductance regulator (CFTR) gene and 60% expressed p63. To further differentiate the cells, the researchers cultured them at an air-liquid interface, and after 5 weeks, the cells expressed several airway and epithelial markers including CFTR (50%) while there was a marked fall in p63 (5%), suggesting that basal epithelial cells had differentiated into mature airway epithelium. Notably, there was a significant increase in airway markers but an absence of expression of DAPT pancreatic, liver and oesophageal markers as well as distal lung markers. This method may therefore be useful to generate epithelium from cystic fibrosis cells from iPS cells. Fibroblasts from patients with CF can be converted to iPS and then differentiated into lung airway epithelium. These cystic fibrosis BML-190 cells from iPS can be used to further investigate the disease. Similarly, other groups have also used developmental pathways and markers to generate lung epithelium in vitro. Mou et?al. used a combination of Nodal, Wnt-�� catenin and staged inhibition of BMP to generate definitive endoderm.[42] Thereafter, they used BMP4, FGF2 and GSK3iXV (Wnt antagonist) to generate anterior endoderm and lung specification but had a significant expression of CDX2 (a marker of posterior endoderm). They found that TGF-�� antagonism was sufficient to get cells from definitive posterior endoderm to anterior lung endoderm as characterized by Nkx2.1, FoxA2 and Sox2 expressing cells. In addition, Smad-dependent BMP2/4 signalling was critical for Doxorubicin lung specification. The authors then found that a combination of BMP7, Noggin, low Wnt and intermediate levels of RA generated proximal airway progenitors (Nkx2.1+, Sox2+), while BMP4, FGF2, FGF7, Wnt 2/2a and Wnt 3 as well as low levels of RA generated multipotent distal lung progenitors (Nkx2.1+, Sox9+) or (Nkx2.1+, Foxp2+) cells. They injected these cells subcutaneously and generated cellular spheres that expressed lung markers. They used a similar patterning protocol on human iPS and found Nkx2.1+ as well as p63+ cells. No further differentiation of the proximal airway was detected. Of note, these protocols could generate distal lung epithelium at 85�C90% efficiency and anterior endoderm at 50�C60%, but well-differentiated cells, characterized by co-expression of a combination of specific markers were much lower (