Efficient Generation of Cynomolgus Monkey Induced Pluripotent Stem Cell-Derived Intestinal Organoids with Pharmacokinetic Functions

Abstract

In preclinical studies, the cynomolgus monkey (CM) model is frequently used to predict the pharmacokinetics of drugs in the human small intestine, because of its evolutionary closeness to humans. Intestinal organoids that mimic the intestinal tissue have attracted attention in regenerative medicine and drug development. In this study, we generated intestinal organoids from CM induced pluripotent stem (CMiPS) cells and analyzed their pharmacokinetic functions. CMiPS cells were induced into the hindgut; then, the cells were seeded on microfabricated culture vessel plates to form spheroids. The resulting floating spheroids were differentiated into intestinal organoids in a medium containing small-molecule compounds. The mRNA expression of intestinal markers and pharmacokinetic-related genes was markedly increased in the presence of small-molecule compounds. The organoids possessed a polarized epithelium and contained various cells constituting small intestinal tissues. The intestinal organoids formed functional tight junctions and expressed drug transporter proteins. In addition, in the organoids generated, cytochrome P450 3A8 (CYP3A8) activity was inhibited by the specific inhibitor ketoconazole and was induced by rifampicin. Therefore, in the present work, we successfully generated intestinal organoids, with pharmacokinetic functions, from CMiPS cells using small-molecule compounds.

Application of Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids as a Model of Epithelial Damage and Fibrosis in Inflammatory Bowel Disease

“…Characteristics of HiOs Previously, we succeeded in generating human and cynomolgus monkey iPS cell-derived intestinal organoids with pharmacokinetic function. 27,28) In this study, we generated HiOs using our method to induce intestinal differentiation. The HiOs formed highly uniform bubble-like structures (Fig.…”

“…32,33) The intestinal organoids offer new possibilities to develop intestinal disease models and perform drug screening with in vivo physiology. 19,20,34) In addition, our differentiation protocol is more useful for application in high-throughput drug screening than conventional methods, 24,35) because we previously established a HiO suspension method to enable mass culture 27) (Fig. 1).…”

“…Human iPS Cell Differentiation into Intestinal Organoids A human iPS cell line (Windy) was cultured as previously reported. 27) HiOs were generated from human iPS cells as reported by us earlier. 27) Briefly, human iPS cells were passaged onto Matrigel-coated plates and cultured with StemSure ® hPSC medium supplemented with 35 ng/mL FGF2 before differentiation.…”

“…27) HiOs were generated from human iPS cells as reported by us earlier. 27) Briefly, human iPS cells were passaged onto Matrigel-coated plates and cultured with StemSure ® hPSC medium supplemented with 35 ng/mL FGF2 before differentiation. After the cells reached 80% confluence, they were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 100 ng/mL activin A for 3 d. The FBS concentration was gradually increased from 0 to 0.2 or 2.0%.…”

The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update

“…These cells were induced into the hindgut then populated on microfabricated culture vessel plates to generate spheroids that were differentiated into intestinal organoids in a medium encompassing small-molecule compounds.The generated organoids were also characterized by a polarized epithelium and were composed by numerous cells that made up small intestinal tissues. Interestingly they also expressed functional tight junctions drug transporter protein, which allows a broader biochemical investigation [ 45 ].…”