We utilised a genetically modified mESC line, Mixl1GFP/w [17], in which sequences encoding GFP were inserted into one allele of Mixl1, a gene whose expression is restricted to the mesendodermal precursors of the primitive streak [21], [22].
This mESC line was modified to express GFP in Lhx6+ cells, a marker of newly postmitotic and mature MGE-derived cortical interneurons.
These random genetic insertion-free mESC lines can be directed to differentiate into a homogenous population of functional ATIICs, providing a reliable source of cells for further characterization of ESC-derived ATIICs in mice lung injury models.
Undifferentiated mESC lines were maintained and expanded in mESC media on feeder layers.
Analysis included samples for each mESC line from at least 2 independent experiments, for all time points, and combined results from mESC lines of similar sex/genotype combinations.
To determine if our approach could be extended to other mESC lines and for long term cultures, we initiated culture of another established line of mESCs (W4, 129/SvEv).
We performed metaphase spreads to karyotype our mESC lines as follows.
To derive mESC lines, Shp2+/−: GFP mice were intercrossed, and blastocysts (E3.5) were collected and cultured as described previously [28].
However, the wt mES cells, like other mESC lines available, showed approximately 5 10% spontaneous differentiation under the same culture condition.
Samples collected on days 0 35 of in vitro differentiation from mESC lines were analyzed as above, from wildtype and Dazl−/− mutant lines.
