Figure 4.

The progeny of a single IPF mesenchymal progenitor cell displays the IPF fibroblast phenotype. A: Derivation of progeny from a single progenitor. Upper panel: SSEA4⁺ cells (99% pure) that had undergone FACS were seeded at one cell per well in 96-well dishes coated with methylcellulose and were observed for colony formation. The arrow denotes a single SSEA4 expressing IPF mesenchymal progenitor cell (MPC). Middle panel: Shown is a phase-contrast image of a colony formed by the progeny of a single progenitor cell. Bottom panel: Expansion of the colony on a tissue culture dish. B: IPF clonal progeny have the biochemical hallmarks of IPF fibroblasts. Western blots of the progeny of mesenchymal progenitors with each lane representing the clonal progeny of a single progenitor, designated IPF clone 1 (lane 3) and IPF clone 2 (lane 4) for collagen 1A1, α-SMA, GAPDH, p-Akt, PTEN, and Cav-1. Results for two primary control mesenchymal cell lines (designated control 1 and 2; lanes 1 and 2) and two primary IPF mesenchymal cell lines (designated IPF 1 and 2; lanes 5 and 6) are shown as a reference. C: IPF clonal progeny form a fibrotic reticulum in zebrafish. An example representative of seven zebrafish embryos grafted with the progeny of a single IPF progenitor. Graft forming a fibrotic reticulum (red) appears in head region with nonspecific autofluorescence outlining the yolk sac (left panel). A representative three-dimensional computer-generated reconstruction of the xenograft 48 hours after grafting shows the formation of a fibrotic reticulum (right panel). D: Quantification of aggregated length of all processes in IPF and control clonal progeny grafts. Data are expressed as follows: the horizontal line in the middle of each box indicates the median, and the top and bottom borders of the box mark the 75th and 25th percentiles; the whiskers above and below the box mark the 90th and 10th percentiles (D). n = 10 (D, IPF grafts); n = 25 (D, control). P < 0.001 (D). Scale bars: 10 μm (A). Cav-1, caveolin-1; Collagen 1A1, type I collagen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-Akt, phospho-Akt; α-SMA, α-smooth muscle actin.