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. 2013 Oct;10(5):S45–S97. doi: 10.1513/AnnalsATS.201304-090AW

Figure 2.

Figure 2.

Alveolar differentiation repertoire of embryonic stem cell (ESC)-derived Nkx2–1+ lung progenitors. (A and B) Immunostaining for alveolar epithelial markers T1α, pro-surfactant protein C (SPC), and Nkx2–1 on cells at the completion of the 25-day directed differentiation protocol. ESCs sorted on Day 15 based on Nkx2–1GFP+ expression gave rise to cells reminiscent of type 1 alveolar epithelial cells (AEC1) as they lost Nkx2–1 nuclear protein expression (green immunostain) while expressing T1α protein (A). (B) Other patches of cells appeared more reminiscent of distal SPC+ alveolar epithelial cells because they expressed punctate cytoplasmic pro-SPC protein and displayed SPC promoter activation while retaining Nkx2–1GFP expression. Arrow = SPC-dsRed and Nkx2–1GFP coexpressing cell (orange). Arrowhead = cell expressing only Nkx2–1GFP. (C) Schematic summarizing the decellularization-recellularization assay. (D) Hematoxylin and eosin stains of lung sections showing lung scaffold appearance with no recellularization (left panel) versus hypercellular sheets after reseeding with undifferentiated ESCs (middle panel) versus cells of alveolar structural morphologies after seeding with Nkx2–1GFP+ purified ESC-derived progenitors (right panel). Scale bars = 100 μm in three left panels. Zoom of the indicated boxed region is shown in far right panel with scale bar = 20 μm. (E and F) Nkx2–1+ nuclear protein (brown; arrowheads in E) immunostaining of engrafted cuboidal epithelial cells in the corners of alveoli derived 10 days after reseeding with Nkx2–1GFP+ sorted cells. Arrow = flattened nucleus of an Nkx2–1 negative cell (purple) lining the alveolar septum. Many of these flattened cells were T1α+ (F; arrowhead). Scale bars = 20 μm. (G) Control mouse lung without decellularization showing T1α apical membrane staining pattern (brown) of mature AEC1 (see Figure S7 in the online supplement). (H) Ciliated airway epithelial cell (arrow) 10 days after reseeding with differentiated/unsorted ESC-derived cells. Scale bar = 20 μm. All nuclei were counterstained with hematoxylin (purple). Adapted by permission from Reference 104.