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In vitro maturation of intestinal organoids differentiated from wild type and STAT3 knock out human embryonic stem cells

ABSTRACT: Human intestinal organoids (hIOs) resemble the human intestine physiologically and structurally. We recently present an in vitro maturation technique for generating mature and functional hIOs from human pluripotent stem cells (hPSCs). Here, we investigated the mechanisms of STAT3 for inducing in vitro maturation of hIOs. Using CRISPR/Cas9-mediated gene editing, STAT3 knockout (KO) human embryonic stem cell (hESC) lines were generated and characterized. By genome-wide microarray analyses, STAT3 KO hIOs showed markedly different profiles from the in vitro matured hIOs and human small intestine and the majority of genes, which are associated with intestinal development and functions, were downregulated by STAT3 KO. This study reveals important signaling pathways for the maturation of hIOs derived from hPSCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE132561 | GEO | 2019/09/30

REPOSITORIES: GEO

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Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

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In vitro maturation of intestinal organoids differentiated from wild type and STAT3 knock out human embryonic stem cells

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