Mice

Inbred FVB/N(H-2q) and transgenic FVB/NCrl-Tg(Pgk1-EGFP)01Narl mice were purchased from National Laboratory Animal Center (Taipei, Taiwan) at 6–8 weeks old. Animals were housed in Animal Care Facility at Chang Gung Memorial Hospital (CGMH) under the standard guidelines from "Guide for the Care and Use of Laboratory Animals" and with the approval of CGMH Committee on Animal Research. Females were caged with males in the afternoon and checked for vaginal plugs the following morning. The day of the plug observed was designated as day 0 of the pregnancy.

Mass culture of dispersed enterocytes

The gut was harvested from FVB/NCrl-Tg(Pgk1-EGFP)01Narl murine fetuses after cesarean delivery on gestational day 14. Fetal gut was treated with 1 mg/ml collagenase/dispase (Roche, Mannheim, Germany) in phosphate-buffered saline (PBS) for 15–30 min at 37 °C. Digested tissue was triturated and washed. Then, dispersed enterocytes at the dose of 1–2 × 106 were seeded into tissue culture dishes (internal ∅87 mm TPP, 93,100) filled with self-renewal medium (SRM) [14,15,16,17]. SRM (100 ml) consisted of 50 ml low-glucose Dulbecco's modified Eagle medium, 30 ml neurobasal medium (Gibco), 15 ml chicken embryo extract, 100 µl retinoic acid (117 µM), 1 ml penicillin–streptomycin, 1 ml N2 supplement (Gibco), 2 ml B27 supplement (Gibco), 100 μl 2-mercaptoethanol (50 mM), basic fibroblast growth factor (20 ng/ml; Peprotech, London, UK) and insulin-like growth factor 1 (20 ng/ml; Peprotech). Cells were passaged every 4–5 days to lessen background mesenchymal cells. Then, enteric neurospheres were collected for transplantation.

Flow cytometric analyses for enteric neurospheres [18]

Enteric neurospheres were dissociated by Accutase (STEMCELL technologies) and subjected to intracellular staining. After fixed, permeabilized and washed with BD Cytofix/Cytoperm™ Kit (BD Biosciences), cells were incubated with primary antibodies against p75 neurotrophin receptor (p75, ab8875, Abcam) and tubulin β3 (TUBB3, TUJ1, BioLegend), followed by fluorescence-conjugated secondary antibodies against the species the primary antibodies were raised in. After vigorous washes, cells were further treated with fluorescence-conjugated anti-S100 calcium-binding protein B (S100b, C-3, Santa Cruz Biotechnology). Finally, cells were acquired by BD FACSCantoTM II and analyzed with BD FACSDiva software.

Ex vivo migration of neurosphere cells on gut explants

Adult murine colon of 2–3 cm was harvested, longitudinally cut open at the mesenteric side and washed with povidone-iodine and saline. Specimens were flattened with mucosa-side up on coverslips in the culture dish and mechanically denuded of mucosa by gentle curettages to expose muscularis. Adult gut explants were irrigated with PBS and finally rinsed with SRM. Fetal gut was obtained from gestational day 14 murine fetuses and cut into pieces of ≤ 5 mm in length on coverslips in the dish. Green fluorescence protein-positive (GFP+) neurospheres were implanted on the denuded muscularis surface of adult gut explant or at one end of fetal gut explant. The dishes were kept stationary for a few minutes to allow neurosphere attachment and then gently filled with SRM to flood the sample slides. The organotypic culture was held at 37 °C in a humidified, 5% CO2-containing incubator with SRM replacement every 3–4 days.

Trans-anal ENSC transplantation

Under anesthesia, the anorectum of adult FVB/N mouse (5 for each gender, 8–12 weeks old) was disinfected using povidone-iodine. Recipient’s rectal mucosa was transfixed by three stay sutures of 6–0 PDS (taper point needle, ETHICON) near anorectal junction at 2, 6 and 10 o’clock positions and then artificially prolapsed by gentle traction of stay sutures. Neurospheres dissociated by Accutase were circumferentially injected into rectal submucosa at a dose of 1–2.5 × 106 [1] in 50 μl saline (15–20 μl in each region), using an Ultra-Fine II insulin syringe, 31-G short needle (Becton, Dickinson and Company). Prolapsed rectal mucosa spontaneously returned after removal of stay sutures. After transplantation, mice were kept on warm blanket till waking up.

Immunohistochemical staining of colon tissue sections for donor ENSC engraftment

The harvested colon specimens of recipients were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tissue sections were deparaffinized, rehydrated and then subjected to heat-induced antigen retrieval. Endogenous peroxidase was eliminated by hydrogen peroxide block (Thermo Fisher Scientific) for 15 min. The sections were permeabilized with Tween-20 and then blocked with 1% BSA. For immunohistochemical staining, tissue sections were first treated with rabbit polyclonal anti-GFP primary antibody (1:200, Cat No. GTX113617, GeneTex) for 1.5 h, followed by biotinylated goat anti-rabbit (rabbit specific HRP/DAB (ABC) detection IHC kit, Abcam) secondary antibody at room temperature for 10 min and then streptavidin-HRP in PBS for 10 min. Color was developed by adding chromogen substrate of DAB solution for 15 min. Finally, the sections were counterstained with hematoxylin for 5 min. The sample slides of the entire colon fixed in a Swiss roll fashion were specifically scanned and converted into high-definition digital data by NanoZoomer 2.0-RS. Images were analyzed by NDP.view2 viewing software for the histological locations of donor cell engraftment.

Chimerism levels of donor ganglia within myenteric plexuses of recipients’ colorectum

The ganglia of myenteric plexuses in immunohistochemically-stained colorectum were enumerated under a 20 × objective. The levels of donor ganglion chimerism were determined by the number of GFP+ ganglia divided by the total ganglion number within the myenteric plexuses of the colorectum specimens.

Immunofluorescence staining of colon tissue sections for enteric ganglia

Formalin-fixed, paraffin-embedded tissue sections were subjected to deparaffinization, rehydration, antigen retrieval and permeabilization as described above. Samples were first incubated with primary antibodies against GFP, followed by fluorescence-conjugated donkey anti-rabbit IgG (Poly4064, BioLegend), and finally treated with anti-TUBB3 and anti-glial fibrillary acidic protein (GFAP, 2E1.E9, BioLegend) antibodies directly conjugated with fluorescence. Visualization of the nuclei was achieved by Hoechst 33,342 staining (1: 20,000, Invitrogen). Sections were mounted with Dako fluorescence mounting medium. Images were taken using a Leica TCS SP8X confocal microscope.

Statistical analyses

All numerical data were shown in boxplots. The equality of means was examined by one-way analysis of variance (ANOVA) among three groups with post hoc Fisher’s least significant difference (LSD) multiple comparisons. Differences were regarded as statistically significant at p < 0.05.