Special AT-rich sequence-binding protein 2 (SATB2) represses proximal small intestine identity in human pluripotent stem cell derived ileal and colonic organoids

Abstract

Background: Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Human intestinal organoids can be generated from human pluripotent stem cells and patterned into distinct intestinal regions including duodenum, ileum and colon. Ileal and colonic organoids express epithelial Special AT-rich sequence-binding protein 2 (SATB2) and generate cell types specific to distinct intestinal regions. SATB2 is the earliest known transcription factor to be specifically expressed in the developing ileal and colonic epithelium in mice. However, the role of SATB2 in regional specification of ileal and colonic organoids has not been determined. The objective of this study was to determine if SATB2 is necessary for establishment of ileal and colonic epithelial identity in organoids. We hypothesized that SATB2 establishes ileal and colonic identity through repression of proximal intestine gene regulatory networks and activation of ileal/colonic gene regulatory networks. Methods: We tested our hypothesis using human induced pluripotent stem cell (IPSC) derived ileal and colonic organoids (HCOs). SATB2 deficient IPSCs were generated using CRISPR-Cas9 mediated gene editing. Duodenal, ileal and colonic organoids were generated as previously described. Human intestinal, ileal and colonic organoids were analyzed using standard biochemical and molecular biology techniques. The effect of SATB2 loss-of-function on the transcriptomes of ileal and colonic organoids was assessed using RNA-seq analysis. Results: Analysis of transplanted duodenal and ileal HCOs revealed that SATB2 deficient ileal organoids displayed a transcriptional profile that was similar to duodenal organoids. SATB2 deficient ileal organoids generated enterocytes and enteroendocrine cell subtypes that are normally expressed in the developing human duodenum. Furthermore, in vitro studies revealed SATB2 deficient HCOs transcriptionally resembled proximal small intestine organoids. SATB2 deficient HCOs did not display altered expression of transcription factors expressed in colonic mesenchyme, consistent with SATB2 expression being confined to the epithelial cells of the organoids. Conclusions: SATB2 is necessary for establishment of ileal and colonic epithelial identity in organoids. SATB2 is able to repress the expression of proximal small intestine genes and activate the expression of ileal and colonic genes. Future studies will examine the mechanism by which SATB2 establishes ileal and colonic identity. These studies will provide important insights in The Múnera lab is funded by NIH/NCI 5U54CA210962-02 South Carolina Cancer Disparities Research Center (SC CADRE), NIH/NIGMS P20 GM130457-01A1 COBRE in Digestive and Liver Disease, NIH/NIDDK 1P30 DK123704-01 MUSC Digestive Disease Research Core Center and NIH/NIDDK R56DK129575. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.