Fig. 4.

Evidence for highly multipotent osteogenic CNC progenitors in clonal cultures. Clonal cultures of CNCCs grown with and without Shh (d7–d10) were analyzed by in situ hybridization and immunocytochemistry to detect osteoblasts, chondrocytes, myofibroblasts, melanocytes, neurons, and glial cells (see Materials and Methods). (A) Quantification of the clones containing each phenotype. Nearly all colonies include osteoblasts; Shh increases the total frequency of the clones containing chondrocytes and myofibroblasts. Data are expressed as mean percent ± SEM from 4 independent experiments (n = 109 and n = 146 colonies in control and Shh-supplemented media, respectively; **, P < 0.01; *, P < 0.05). (B–E) GNMFCO multiphenotypic colony including: (B) Runx2⁺osteoblasts, (C) α-SMA⁺ myofibroblasts (red) and HNK1⁺ glial cells (green), (D) neurons (tyrosine hydroxylase-tubulinβIII⁺ in red, indicated by the arrow next to the glial cells), and (E) melanocytic cells (MelEM⁺ in green, next to the neurons). Asterisks in B–D indicate cartilage nodules. (Magnification: 140×.)