This paper describes a novel technique to engraft human colon organoids in vivo orthotopically, which allowed the authors to study human colon stem cell functionality.
It was observed that complete removal of the host epithelium (NOD.Cg‑PrkdcscidIl2rgtm1Sug/Jic (NOG) mice) was required for long-term stable engraftment. Damage was induced via hot EDTA treatment and mechanical scraping of one side of the colon mucosa.
Engraftment of GFP expressing organoids following damage had an average success rate of 79.5% (n=3), and GFP+ areas could be detected for over 6 months via endoscopy, which was confirmed to be human via the expression of human specific cytokeratin.
The engrafted human organoid tissue formed large crypts in vivo, which were more comparable with the size of human crypts than mouse. In addition, the pattern of goblet cell mucin expression, which differs in human and mice, was consistent with that observed in human tissue as opposed to mouse. These data suggest the organoid tissue harbored cell-intrinsic controls for the determination of size and mucin type.
Lineage tracing experiments of human colon Lgr5+ stem cells revealed that cells took 10 days to migrate to the top of the crypt. Ribbon-like structures typically seen when tracing stem cells were observed after 28 days, and nearly all crypts were monoclonal by day 180. Progeny were observed to have differentiated into all intestinal lineages. Therefore, the engrafted human Lgr5+ stem cells maintained their self-renewal and differentiation capabilities in vivo.
The proliferative properties of the human and mouse Lgr5+ stem cells in vivo were compared by incorporation of EdU and Ki67 expression. The mouse Lgr5+ stem cells were highly proliferative, with 90% Ki67+ and 30% EdU+. In comparison, Ki67 and EdU expression were significantly lower in the human Lgr5+ stem cells suggesting that the human stem cells cycled slower than the mouse stem cells, even whilst in the same tissue environment.
This paper describes a new model that could allow further investigation into human intestinal stem cells within an in vivo setting. In addition, no tumorigenesis was observed over 10 months of engraftment, suggesting the organoid tissue maintained genetic stability, supporting the use of human organoid tissue in cell therapy / regenerative medicine.