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Genome-wide epigenetic and transcriptomic characterisation of human-induced pluripotent stem cell-derived intestinal epithelial organoids

Human intestinal epithelial organoids (IEOs) are self-organising, three-dimensional structures that can be propagated long term in culture and be differentiated into numerous epithelial cell subsets that are present in gut tissue. They have become an important in vitro tool to study key intestinal properties, responses to environmental assaults and drug therapies, without the requirement for invasive procedures to obtain human tissues. IEOs can be generated either from embryonic or adult intestinal pluripotent stem cells (iPS cells) or from iPS cells created in vitro via retro-virus mediated reprogramming of differentiated human cells e.g. skin cells, to a pluripotent state. While organoids derived from Lgr5+ adult stem cells give rise to cultures that exclusively consist of intestinal epithelial cells, pluripotent stem cell-derived organoids may also contain mesenchymal cells. Recent developments in culturing protocols have also allowed us to generate epithelial cell-specific intestinal organoids from iPS cells. A pure epithelial organoid model offers unique opportunities to study the cells' intrinsic and cell-type-specific mechanisms as they mimic in vivo structure and the cellular dynamic of the gut from which they were derived or were differentiated into.

In this study the authors aimed to characterise organoid cultures derived from human iPS cells and to compare them with cultures derived from primary intestinal epithelial cells.

Intestinal epithelial organoid cultures were derived from at least three different lines of iPS cells. Cells were differentiated into definite endoderm followed by embedding the posteriorised cells into Matrigel to establish 3D organoid structures. Organoids were also established from primary intestinal epithelial cells obtained from tissue biopsies of sigmoid colon (SC) and terminal ileum (Tl) from consented patients.

Organoid cultures showed comparable morphology highlighted by the presence of cell adhesion molecules and epithelial polarisation.

All organoids derived from Tl, SC and differentiated iPS cells were found to express several epithelial cell markers including E-cadherin (CDH1) and the intestinal stem cell marker LGR5 (Lgr5). They also showed ubiquitous expression of enterocyte marker Villin 1, with specific subset of cells expressing mucin 2, chromogranin A and lysozyme, markers associated with the Goblet, Enteroendocrine and Paneth cells, respectively.

Genome-wide analysis of DNA methylation and transcriptomic profiling (by RNA sequencing) using Illumina platforms revealed relative sample similarities. iPS cell-derived organoids clustered more closely to primary intestinal colonic epithelium purified from mature gut when compared to human fetal proximal and fetal distal gut. Interestingly, iPS cell-derived organoids had a greater number of significantly methylated positions and differentially expressed genes in common with the SC than with Tl, suggesting the iPS organoids culture conditions guided cells to differentiate towards fate of epithelial cells found in the terminal portion of the colon.

In addition, when transcriptomic data obtained from SC-derived organoids was compared to data from iPS cell-derived organoids, focusing on the most significant differentially expressed genes, intriguing differences in gene expression patterns were observed. Some key small intestinal markers for Paneth cells e.g. DEFA5 and DEFA6 were found to be abundant in iPS cell-derived organoids, while genes involved in innate defense (e.g. IFI27, HCP5 or PIGR) showed markedly lower expression levels. These results indicated that iPS cell-derived organoids may have not reached the full level of differentiation into mature intestinal epithelium as found in vivo.

In summary, although human iPS cell-derived organoids provide a useful research model to study functional aspects of human intestinal epithelial pathophysiology and hold a great potential as a translational research tool to investigate new treatments for gastrointestinal diseases, such models need to be further optimised, fully characterised and validated to serve as reliable in vitro GI models that correspond to intestinal development and functionality displayed in vivo.

Reference

Genome-wide epigenetic and transcriptomic characterization of human-induced pluripotent stem cells-derived intestinal epithelial organoids. Kraiczy et al, 2019; Cellular and Molecular Gastroenterology and Hepatology 7(2):285-288

https://doi.org/10.1016/j.jcmgh.2018.10.008

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