Abstract
Human intestinal organoids (hIOs), which resemble the human intestine structurally and physiologically, have emerged as a new modality for the study of the molecular and cellular biology of the intestine in vitro. We recently developed an in vitro maturation technique for generating functional hIOs from human pluripotent stem cells (hPSCs). Here, we investigated the function of STAT3 for inducing in vitro maturation of hIOs. This was accompanied by the tyrosine phosphorylation of STAT3, whereas treatment with pharmacological inhibitors of STAT3 suppressed the phosphorylation of STAT3 and the expression of intestinal maturation markers. We generated and characterized STAT3 knockout (KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9-mediated gene editing. We found that STAT3 KO does not affect the differentiation of hESCs into hIOs but rather affects the in vitro maturation of hIOs. STAT3 KO hIOs displayed immature morphologies with decreased size and reduced budding in hIOs even after in vitro maturation. STAT3 KO hIOs showed markedly different profiles from hIOs matured in vitro and human small intestine. Additionally, STAT3 KO hIOs failed to maintain upon in vivo transplantation. This study reveals a core signaling pathway consisting of STAT3 controlling the in vitro maturation of hIOs derived from hPSCs.
Highlights
Human intestinal organoids are stem cell-derived, three-dimensional (3D) multicellular structures that closely mimic the physiology, functions, and cell organizations of intestinal tissues in vivo [1,2]. hIOs have been previously derived from human pluripotent stem cells or native biopsies, both of which are known to share similar properties, including the specialized intestinal epithelial cell types within hIOs and crypt-villus structures similar to those of the intestinal epithelium [3,4]. hIOs are a promising tunable source for intestinal developmental and disease modeling, drug absorption, and toxicity testing, and gut microbiota interaction models. hPSC-derived hIOs create additional layers of diverse mesenchymal cells that are highly representative of the intestines in vivo [5]
We previously showed that the simulation of the human intestinal environment by co-culturing hIOs derived from human embryonic stem cells and human induced pluripotent stem cells with human T lymphocytes induced the in vitro maturation of hIOs, where interleukin-2 (IL-2) was identified as the major promoting factor of maturation
Transmission Electron Microscopy (TEM) analysis demonstrated that the brush border in Mat-hIO epithelium was well developed and Mat-hIO epithelium had longer microvilli than those of control hIO epithelium (Cont-hIOs) (Figure 1b)
Summary
Human intestinal organoids (hIOs) are stem cell-derived, three-dimensional (3D) multicellular structures that closely mimic the physiology, functions, and cell organizations of intestinal tissues in vivo [1,2]. hIOs have been previously derived from human pluripotent stem cells (hPSCs) or native biopsies, both of which are known to share similar properties, including the specialized intestinal epithelial cell types within hIOs and crypt-villus structures similar to those of the intestinal epithelium [3,4]. hIOs are a promising tunable source for intestinal developmental and disease modeling, drug absorption, and toxicity testing, and gut microbiota interaction models. hPSC-derived hIOs create additional layers of diverse mesenchymal cells that are highly representative of the intestines in vivo [5].hPSC-derived hIOs have several limitations, such as immature, fetal-like transcriptional profiles and limited functionalities [6,7]. HIOs have been previously derived from human pluripotent stem cells (hPSCs) or native biopsies, both of which are known to share similar properties, including the specialized intestinal epithelial cell types within hIOs and crypt-villus structures similar to those of the intestinal epithelium [3,4]. The cellular and functional maturation of hPSC-derived hIOs required in vivo transplantation into immunocompromised mice [8] or in vitro maturation by co-culture with immune cells or by exposure to IL-2 as in our previous study [9]. STAT3 mediates the activation of acquired immune responses, which seem to play a role in the pathogenesis of colitis by promoting the survival of pathogenic T cells [15]. The pharmacological regulators of STAT3 maintaining STAT3 homeostasis and adequate STAT3 levels may be promising therapeutic candidates for intestinal disorders
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