Postnatal intestinal engraftment of prospectively selected enteric neural crest stem cells in a rat model of Hirschsprung disease

Abstract

Identification of neuronal progenitor/stem cells in the postnatal gut suggests the development of transplantation approaches to enteric nervous system (ENS) diseases. Many clinical applications would require engrafting large segments of postnatal gut in vivo. We investigated the ability of unselected gut cells vs selected enteric neural crest stem cells (eNCSCs) to engraft and differentiate in the postnatal gut in the Hirschsprung disease (HD, ednrb(sl/sl)) rat. Total intestinal cells or eNCSCs (α(4) integrin(+), p75(++)) from embryonic day (E)14.5 rats carrying a marker transgene (human placental alkaline phosphatase, hPAP) were injected intraperitoneally (i.p.) into neonatal HD rats and their healthy littermates. The entire gut was systematically analyzed 3 weeks later for hPAP(+) cells between the serosal surface and the muscularis mucosae. Engrafted cells were examined for HuC/D, S-100B, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) expression. No rats (0/33) injected with unselected cells had hPAP(+) cells in the ENS that expressed neuronal or glial markers. 5/11 healthy and 4/5 HD rats injected with eNCSCs showed widespread but low density engraftment in the ENS with cells expressing neuronal or glial markers. Neurons expressed nNOS and VIP. There was no engraftment in the colon of either HD or wildtype rats. Enteric neural crest stem cells will engraft diffusely throughout the postnatal gut of HD rats and differentiate into neurons and glia. Engraftment is not uniform, likely related to age-dependent changes in the gut mesenchyme. Intraperitoneal injection is easily performed in sick neonates and may be developed as a technique to supply exogenous ENS cells to the diseased postnatal gut.