Alcian blue

For chondrogenic differentiation, hDPSCs from four donors were seeded at a density of 8 × 10³ cells per well in a 6-well plate and cultured in α-MEM complete medium. When the hDPSCs reached 90% confluence, the cultured medium was removed, and the cell pellet was maintained in hMSC chondrogenesis induction medium, serum-free kit (Provitro, Berlin, Germany). hDPSCs were cultured for 28 days in a chondrogenesis induction basal medium containing dexamethasone, sodium pyruvate, ascorbic-acid-2-phosphate, proline and antibiotics, which was supplemented by the human transforming growth factor-beta 3 (TGF-β-3). Growth factors were added fresh daily in concentration 10 µL/mL induction medium. The culture medium was changed twice a week. The status of the differentiated hDPSCs was confirmed using Alcian Blue (Abcam, Inc, Cambridge, UK) staining. After 28 days of culture, the cells were washed with PBS, fixed in 10% formalin solution (Merck, Saint Louis, MO, USA), washed in PBS again and incubated in the Alcian Blue solution for 30 min. In the next step, the dishes with the differentiated cells were washed once in running tap water and twice in distilled water [50 (link)]. The dishes with the stained cells were analyzed using an Olympus IX73 microscope (Olympus, Tokyo, Japan).

Six-micrometer sections of both eyes and the adnexa were stained for mucin with Alcian blue, pH 2.5, using a kit (Abcam Inc, Cambridge, MA). The eye sections from each group were assessed in a 0.1-mm² area of either the cornea or inferior fornices of the conjunctiva. The stained sections were then examined and imaged using a virtual microscope (NanoZoomer 2.0 RS, Hamamatsu, Japan).

The recipient colon tissues were isolated on 14-day PT, fixed with 10% formalin (Sigma-Aldrich, MO, USA), and incubated with 15–30% sucrose for cryopreservation. Xenograft colons were embedded in Tissue-Tek O.C.T. (Sakura Finetek, Cat No. 4583, Sakura Finetek, CA, USA) compound and sectioned at 10 μm. The tissues were stained with haematoxylin and eosin for histological analysis. For histopathological analysis, Alcian Blue (Abcam, Cat No. 150662, Abcam, Cambridge, UK)-Periodic Acid Schiff (Sigma-aldrich, Cat No. 1.01646.0001) staining was performed on 10-μm colon sections. AB-PAS-stained sections confirmed mucus-secreting goblet cells and mucin. Sections were observed using a microscope (BX53; Olympus, Tokyo, Japan). The colon crypt depth was measured in H&E-stained images using Image J software (Matrigel group, n = 675 crypts; ISCs^3D-hIO group, n = 705 crypts).

Isolated intestine was flushed with ice-cold 1X phosphate-buffered saline (PBS) and fixed in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature overnight, then transferred into 70% ethanol, processed with a tissue processor (Excelsior AS Tissue Processor; Thermo Fisher Scientific), followed by embedding in paraffin and then cutting into 5 μm sections.
For H&E staining, paraffin sections of the intestine were stained with H&E by following the manufacturer's protocol (Sigma-Aldrich) and analyzed under a bright-field microscope.
For Alcian Blue staining, paraffin sections of the intestine were stained with an Alcian Blue Kit (Abcam) by following the manufacturer's protocol. The blue goblet cells in the crypt and villus were counted visually or analyzed by using Fiji ImageJ to count the positive cells.