Intestinal stem cells contribute to the maturation of the neonatal small intestine and colon independently of digestive activity

The murine intestine, like that of other mammalians, continues to develop after birth until weaning; however, whether this occurs in response to an intrinsic developmental program or food intake remains unclear. Here, we report a novel system for the allotransplantation of small intestine and colon harvested from Lgr5 EGFP-IRES-CreERT2/+; Rosa26 rbw/+ mice immediately after birth into the subrenal capsule of wild-type mice. By histological and immunohistochemical analysis, the developmental process of transplanted small intestine and colon was shown to be comparable with that of the native tissues: mature intestines equipped with all cell types were formed, indicating that these organs do not require food intake for development. The intestinal stem cells in transplanted tissues were shown to self-renew and produce progeny, resulting in the descendants of the stem cells occupying the crypt-villus unit of the small intestine or the whole crypt of the colon. Collectively, these findings indicate that neonatal intestine development follows an intrinsic program even in the absence of food stimuli.


Supplementary Figures
Supplementary Figure 1. Maturation of neonatal small intestine to possess features comparable to the adult intestine. (a, a', e, e', i, i', m, m') H&E staining for small intestine at the indicated birth date. (b, b', f, f', j, j', n, n') Immunostaining for lysozyme (red, Lys) on sections prepared from Lgr5 CreERT2/+ mice at the indicated birth date (green, Lgr5).
(c, g, k, o) Periodic acid-Schiff (PAS) staining for the small intestine at the indicated birth date. (d, h, l, p) Alkaline phosphatase staining for the small intestine at the indicated birth date.
(q, q') Immunostatining of intestinal tissues for the brush border enzyme sucrose-isomaltase (SIM) (q'; arrow) was used to assess functional maturation.
Error bars indicate standard deviation. **; p < 0.001, *; p < 0.01. segmented single-colored areas were observed in the horizontal sections of the lower villi. Because each single-colored area can be considered derived from stem cells, it was suggested that P7 stem cells located at the crypt-base had started to supply their desendants to occupy the lower part of the villi. Multiple single-colored areas suggested that there existed several stem cells in the single crypt and therefore the lower villi were polyclonal. However, the difference in color arrangement was observed between the stem-cell derived multiple clones observed in lower villi and mingled pattern in upper villi. The gray line indicates the boundary between these two color patterns. To observe the developmental process of intestines under the environment without exposure to nutrient and digestive activity, the neonatal small intestine was harvested from Lgr5 EGFP-IRES-CreERT2/+ mice and transplanted into the kidney subrenal capsule of 8-week-old C57BL/6 recipients.

Supplementary
The transplanted small intestine developed in a comparable manner with naïve counterparts. P; postnatal day. (a-a") Small intestine (a') and proximal colon (a") tissues were dissected from P0 Lgr5 EGFP-Ires-CreERT2/+ mice (a). The length of the cut tissues was 2mm (longitudinal).  Higher magnification images (a', b', h', i', o', p') of the corresponding boxed area are shown below the respective images. White dashed lines indicate the boundary between epithelium and lamina propria. Scale bars: orange, 50 μm; red, 20 μm; white, 10 μm. Figure 9. Subrenal capsule can support the development of transplanted neonatal colon to complete maturation. (a, a', f, f', k, k', p, p', u, u') H&E staining for transplanted colon derived from P0 Lgr5 EGFP-IRES-CreERT2/+ mice at the indicated days after transplantation. (b, g, l, q, v) Immunostaining of GFP (brown) to detect the expression of Lgr5 using colon of Lgr5 EGFP-IRES-CreERT2/+ mice at the indicated days after transplantation.  (a) Developmental process of small intestine; our results indicated that intestine from P0 to P7 exhibited neonatal-type feature, whereas that after P7 showed features comparable with those of an adult.

Supplementary
(b) Representative image of the Fetal Enterospheres (FEnS), which had been expanded after an 8day long culture, from epithelial cells harvested from the proximal regions of the small intestine in P0 C57BL/6 mice.
(c, d, e) Representative images of the organoids, which had been expanded after an 8-day long culture, from the crypts harvested from proximal regions of the small intestine in P7 (c), P14 (d), and P56 (e) C57BL/6 mice. The features of the organoids rather than spherical shape demonstrated that these organoids were derived from matured crypts. (f) The emergence rate of FEnS and organoids from the proximal regions of the small intestine at the indicated birth dates. P0; n = 3, P7; n = 2, P14; n = 3.
(g) The emergence rate of FEnS and organoids from the transplanted proximal regions of the P0 small intestine at the indicated days after transplantation. Day 7; n = 5, Day 14; n = 5. (h-o') Grafts were harvested at 7 days (h-k) or 14 days (l-o') after transplantation and their crypts were served for in vitro culture. Formed organoids at day 8 were analyzed.