Figure 6.

Enrichment of enteric nervous system (ENS) cells from human colon cell cultures, and behavior in embryonic intestine explants. (A) p75⁺ cells could be isolated from colon monolayer cultures and comprised less than 5% of the population sorted by flow cytometry. (B) Neural crest (NC) gene expression levels after quantitative reverse transcription and polymerase chain reaction (qRT-PCR) of FOXD3, SOX10, AP2, and p75 mRNA levels. Groups compared were unsorted colon cells analyzed prior to cell culture (PC), cells that were cultured and analyzed prior to sorting (PS), and cells that were sorted for p75⁺ (p75) and p75 negatively sorted (NS). Box plots show the first quartile to interquartile range, whiskers show minimum and maximum range, and the median is represented by horizontal line. *P < .05; ***P < .01. (C) p75-sorted cells displayed neuronal (white arrowhead) and multipolar morphologies in vitro. (D) p75-sorted cells formed aggregates after centrifugation. (E) p75⁺ aggregates stained for SOX10 and Hu. Inset image is a cell aggregate of unsorted cells (PS) that underwent spin aggregation. In this case, ENS cells are a minor proportion of the total cell population. Data derived from six HSCR patients for flow cytometry study, four Hirschsprung disease (HSCR) patients for qRT-PCR analysis, and three HSCR patients for immunohistochemical study. (F) Human p75⁺ cells migrate distally in aneural quail embryo gut (gut border indicated by dotted white line) (four explants) from the initial placement of the p75⁺ cell aggregate (dotted yellow line). The migrating p75⁺ cells coexpressed SOX10. The direction of migration proximal to distal is indicated by the white arrow. Images acquired by confocal microscopy.