Figure 5. Transplanted Ednrb^sl/sl fetal gut neural crest progenitors form neurons in the aganglionic region of fetal Ednrb^sl/sl guts.

p75⁺α ₄⁺ gut NCSCs were isolated by flow-cytometry from E14.5 Ednrb^sl/slhPAP⁺ or Ednrb^+/+hPAP⁺ fetuses and expanded in culture for 6 to 12 days. The cells were then injected into the distal colon of Ednrb^sl/sl E14.5 fetal rats. Injected guts were explanted onto the chorioallantoic membrane of 8–10 day old chick embryos and developed for 4–5 days before being sectioned and analyzed. Sections were initially reacted chromogenically to identify hPAP+ cells in rat guts injected with Ednrb^sl/sl (A; purple) or Ednrb^+/+ neural crest progenitors (B; purple). Sections adjacent to those containing engrafted cells were then stained with antibodies against hPAP (green, D, H, L), β III-tubulin (blue, E, I, M) and NPY (F), nNOS (J), or VIP (N) and analyzed by confocal microscopy. Panels D–G represent the same section. Panels H–K and L–O represent other sections. White arrowheads indicate triple labeled cells. Ednrb^sl/sl and Ednrb^+/+ cells expressed similar markers after engraftment (C). None of the hPAP+ cells expressed GFAP in these experiments. There was no difference in the length of gut engrafted by Ednrb^+/+ or Ednrb^sl/sl cells (P).