A xenogeneic-free system generating functional human gut organoids from pluripotent stem cells

Abstract

Functional intestines are composed of cell types from all 3 primary germ layers and are generated through a highly orchestrated and serial developmental process. Directed differentiation of human pluripotent stem cells (hPSCs) has been shown to yield gut-specific cell types; however, these structures do not reproduce critical functional interactions between cell types of different germ layers. Here, we developed a simple protocol for the generation of mature functional intestinal organoids from hPSCs under xenogeneic-free conditions. The stem cell-derived gut organoids produced here were found to contain distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, that were derived from all 3 germ layers; moreover, they demonstrated intestinal functions, including peptide absorption, and showed innervated bowel movements in response to stimulation with histamine and anticholinergic drugs. Importantly, the gut organoids obtained using this xenogeneic-free system could be stably maintained in culture for prolonged periods and were successfully engrafted in vivo. Our xenogeneic-free approach for generating gut organoids from hPSCs provides a platform for studying human intestinal diseases and for pharmacological testing.

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

“…Cells were treated with ammonia for 2 days to obtain ammonia-selected cells. The ammoniaselected cells were propagated for 7 days after exposure to ammonia in the modified F medium [28] until semi-confluence, and then passaged to 10-cm dishes after trypsinization with 0.25% trypsin/EDTA. In cases that number of cell death after ammonia treatment was high, the medium was changed immediately.…”

Fetal therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells

“…The organoid self-organizes in 3D culture through self-renewal and differentiation capacities of pluripotent stem cells, such as iPSCs and ESCs. Organoids, such as eye, gut, liver, and brain, are being developed (Hasegawa et al, 2016; Sasai, 2013; Takebe et al, 2012; Uchida et al, 2016; Yokobori et al, 2016). A skin organoid, however, has not been developed so far to assure strength to cover the defect site of MMC.…”

Immortalization of human zone I hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

“…PCA, a multivariate analysis technique which finds major patterns in data variability, was performed to categorized HepaMN cells into each stage of ESCs undergoing hepatic differentiation. The differentiation protocol was previously reported in detail (18).…”