Stochastic clonal expansion of "superstars" enhances the reserve capacity of enteric nervous system precursor cells.

We quantified cell population increase in the quail embryo enteric nervous system (ENS) from E2.5 (about 1500 cells) to E12 (about 8 million cells). We then probed ENS proliferative capacity by grafting to the chorio-allantoic membrane large (600 cells) and small (40 cells) populations of enteric neural crest (ENC) cells with aneural gut. This demonstrated that ENC cells show an extremely high capacity to regulate their proliferation while forming the ENS. Previous mathematical models and clonal label experiments revealed that a few dominant ENS "superstar" cell clones emerge but most clones are small. The model implied that "superstars" arise stochastically, but the same outcome could arise if "superstars" were pre-determined. We investigated these two modes mathematically and by grafting experiments with large and small numbers of ENCs, each including one EGFP-labelled ENC cell. The stochastic model predicts that the frequency of "superstar" detection increases as the ENC population decreases, the pre-determined model does not. Experimentally, as predicted by the stochastic model, the frequency of "superstar" detection increased with small ENC cell number. We conclude that ENS "superstar" clones achieve this status stochastically. Clonal dominance implies that clonal diversity is greatly reduced and in this case, somatic mutations may affect the phenotype. We suggest that somatic mutations coupled with loss of clonal diversity may contribute to variable penetrance and expressivity in individuals with genetically identical ENS pathologies.

Transserosal migration of enteric neural stem cells: Developing an avian colon model.

BACKGROUND: Stem cell transplantation is a potential therapy for enteric neuropathies, including Hirschsprung disease. Proof-of-principle has been obtained using focal transplants into neonatal mouse colon. The challenge now is to deliver stem cells to a large surface area to reconstruct an enteric nerve plexus. One proposed method is serosal application using a polymer membrane. However, transserosal migration of stem cells has not been demonstrated in mature colon. This study aimed to develop an avian model to demonstrate stem cell migration across the intact serosa of mature colon. METHODS: Hindguts were obtained from E14 quail embryos, transplanted onto E8 chicken chorioallantoic membranes and harvested after 2 and 8 days. Tissues were assessed immunohistologically for apoptosis (caspase-3), maturity (α-SMA), preservation of mucosa (E-cadherin), and preservation of serosa (cytokeratin). RESULTS: Transient necrosis of the central mucosa was observed over the first two days, followed by recovery. Twenty-three grafts were assessed immunohistologically at day 8. Nineteen grafts demonstrated progressive maturation and an intact mucosa. Circumferential serosal preservation was observed in 9 grafts. No apoptosis was seen. CONCLUSION: Avian colon may be successfully harvested with an intact serosa. Large chorioallantoic membrane grafts remain viable for at least 8 days, and the serosa can be preserved throughout. This provides an economical platform for assessing transserosal migration of stem cells in mature colon.