Retinoic Acid Promotes the In Vitro Growth, Patterning and Improves the Cellular Composition of Human Pluripotent Stem-Cell-Derived Intestinal Organoids

Abstract

Human intestinal organoids (HIOs) generated from human pluripotent stem cells hold great promise for modeling human development and as a possible source of tissue for transplantation. HIOs generate all of the main epithelial and mesenchymal cell types found in the developing human intestine and mature into intestinal tissue with crypts and villi following transplantation into immunocompromised mice. However, incomplete in vitro patterning and the presence of contaminating neurons could hinder their use for regenerative medicine in humans. Based on studies in model organisms, we hypothesized that the treatment of HIOs with all trans retinoic acid (ATRA) would improve their in vitro growth and patterning. We found that ATRA not only improved the patterning of HIOs, ATRA also increased organoid forming efficiency, improved epithelial growth, enriched intestinal subepithelial myofibroblasts (ISEMFs) and reduced neuronal contamination in HIOs. Taken together, our studies demonstrate how the manipulation of a single developmental signaling pathway can be used to improve the survival, patterning and cellular composition of HIOs.

Human Pluripotent Stem Cell Derived Organoids Reveal a Role for WNT Signaling in Dorsal-Ventral Patterning of the Hindgut

“…Following definitive endoderm induction, cells were treated for 4 days with FGF4 (500 ng/ml; R&D Systems) and CHIR99021 (3 μM; Stemgent) in RPMI 1640 with 2.0% dFBS to generate 3-dimensional mid-hindgut spheroids. For WNT inhibition and activation studies we used mid/hindgut monolayer that was dissociated into clumps as previously described 50 . These mid/hindgut clumps were embedded in basement-membrane Matrigel (BD Biosciences) and subsequently grown in Advanced DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 μM HEPES, penicillin/streptomycin, and EGF (100 ng/ml; R&D Systems).…”

Host-microbiota interaction in intestinal stem cell homeostasis

“… 172 , 173 In monogastric animals, vitamins produced by the intestinal microbiota are primarily absorbed in the colon, whereas dietary vitamins are absorbed in the proximal small intestine. 174 , 175 Vitamin A and its metabolite, retinoic acid, enhance ISC stemness and promote ISC differentiation, respectively, 176 , 177 whereas vitamin B9 rescues the reduction in cell metabolic activity in small intestinal organoids caused by the chemotherapeutic agent methotrexate. 178 The effects of 1,25-dihydroxyvitamin D3 (vitamin D3) on ISC function are controversial, with studies reporting contrasting impacts.…”

SMAD1 Is Dispensable for CDX2 Induction but Required for the Repression of Ectopic Small-Intestinal Gene Expression in Human-Pluripotent-Stem-Cell-Derived Colonic Organoids

“…2.4. Three-Dimensional Culture and Posterior Patterning of Dissociated Mid/Hindgut Endoderm Clumps were generated from the mid/hindgut endoderm monolayer as detailed previously [18]. Briefly, the monolayer was scraped from the 24-well tissue culture plate using 1000 µL pipet tips.…”