Functional Enterospheres Derived In Vitro from Human Pluripotent Stem Cells

Abstract

SummaryIntestinal organoids derived from human pluripotent stem cells (hPSCs) are valuable in vitro research models that enable simplified access to human gastrointestinal tissues. Here, we report the in vitro generation of enterospheres (hEnS) from hPSC-derived gastrointestinal epithelial precursors. hEnS are cystic spheroids with a simple uniform structure composed entirely of intestinal epithelium. hEnS express markers of mature brush border cells and share a transcriptome profile similar to that of more mature intestinal organoids. Modulation of signaling cues enables control of hEnS growth and differentiation, including long-term propagation. We show that hEnS can be exploited for functional studies: hEnS display an innate immune response when treated with enteric pathogens, and transgenic modification of hEnS with a fluorescence cell-cycle reporter enables hEnS-forming stem cell enrichment. Our work establishes hEnS as an accessible and tractable in vitro modeling system for studying human gastrointestinal biology.

Comparison of human and mouse fetal intestinal tissues reveals differential maturation timelines

“…This work provides new insights into intestinal development and maturation during human gestation and should serve as a guide for other researchers studying the intestine, including hPSC-derived tissues, to gauge the relative maturation status of their samples. Our work underscores the difference in the timeline of intestinal development between humans and mice, and reinforces the utility of ex vivo models using human cells and tissues, such as intestinal organoids to further study intestinal development and maturation in humans (Forster et al, 2014;Hill et al, 2017;Nadkarni et al, 2017;Senger et al, 2018;Spence et al, 2011).…”

“…Our data demonstrates that CHGA is present in the developing epithelium as early as week 13 of gestation, indicating that, similar to mouse, EECs are one of the first differentiated epithelial cell types to emerge in the human intestine Noah et al, 2011). The early emergence of EECs during human intestinal development raises questions as to why EECs are typically absent in intestinal tissues differentiated from hPSCs in vitro (Fordham et al, 2013;Forster et al, 2014;Nadkarni et al, 2017), when other cells types that arise later in development can be found. The lack of EECs could be attributed to deficiencies in exogenous signaling that activate genes responsible for promoting EEC differentiation, such as Neurogenin-3 (Sinagoga et al, 2018;Spence et al, 2011).…”

“…Understanding tissue maturation during development can provide important insights into the processes that can contribute to diseases in early infancy and later adulthood. In addition, it has become increasingly clear that a gap in the understanding of how tissues mature is also impacting the production of cell types, including intestinal cells, from pluripotent stem cells that faithfully recapitulate adult tissue function (Finkbeiner et al, 2015;Fordham et al, 2013;Hill et al, 2017;Nadkarni et al, 2017;Spence et al, 2011). While tracking the expression of protein markers of maturation in fixed tissue sections of human intestine from different gestational ages, we observed differences to the mouse intestine for the expression of markers that have been previously described as hallmarks of maturation Finkbeiner et al, 2015;Patel et al, 2012).…”

“…EECs emerge at around E16.5 in mice shortly after villus morphogenesis begins , but it is unclear how early EECs emerge in the human intestine. EECs are considered a putative indicator of maturation due to the difficulty in generating them in vitro in human pluripotent stem cell (hPSC)-derived intestinal tissues which tend to be fetal-like (Fordham et al, 2013;Forster et al, 2014;Nadkarni et al, 2017). The expression of Chromogranin A (CHGA), a neuroendocrine secretory protein, marks EECs.…”

Generation of intestinal organoids derived from human pluripotent stem cells for drug testing

“…Therefore, further maturation of hiPSC-IECs would be required. Procedures that could be useful include heterotopic transplantation into a kidney capsule 24 , transplantation of intestinal grafts prepared by bioengineering technique 26 , co-culture with intestinal neurons 27 and myocytes, suppression of the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) gene 28,29,30 , and stimulation by diet ingredients.…”