Loss of MIST1 expression improves active ISC function in culture. (A) Representative in situ hybridization of Olfm4 in jejunal tissue from Mist1+/+ and Mist1–/– mice. Scale bar equals 20 μm. (B) Quantification of Olfm4 in situ hybridization staining using ACD scoring system. NS, not significant. (C) Micrographs of enteroid cultures derived from jejunal crypts from Mist1+/+, Mist1–/– and Atoh1–/– mice 1, 3, and 6 days after culture. (D) Quantification of budding percentage of Mist1+/+, Mist1–/– and Atoh1–/– enteroids over time. n = 5 Mist1+/+ and Mist1–/– mice were used for 4 independent culture experiments. n = 2 Atoh1–/– mice for 2 independent culture experiments to verify the inability of Atoh–/– crypts to grow in minimal culture medium. For each experiment n = 6 separate wells from each mouse were used for quantifying budding. **P <.01. ***P <.001.