Development of self-renewing 3D organoid culture from human fetal biliary tree stem cells (hBTSCs) as a potential system for regenerative medicine and disease modelling

Background and Aims: 3D organoids represent an advanced culture technology in the field of stem cells and regenerative medicine, recapitulating embryonic development and the physiology of the tissue of origin. Adult or fetal biliary tree represent ideal cell sources of stem/progenitor cells to be used for the regenerative medicine of liver and pancreas. The aim of our study was to generate 3D organoid cultures of hBTSCs suitable for regenerative medicine of liver and pancreas and for in vitro disease modeling Method: The fetal biliary tree (n=6, obtained from elective pregnancy termination) was digested, mechanically and enzymatically, to isolate EpCAM/LGR5-enriched hBTSCs. Cells were then embedded in Matrigel and cultured in an expansion organoid medium containing soluble factors typical of the stem cell niche including EGF, FGF, Noggin, R-Spondin1; these factors represent LGR5 ligands and Wnt agonists and favor the expansion of stem cells and maintenance of stemness. Culture medium was also supplemented with Forskolin, a cAMP activator, and with a TGFβR inhibitor, A83-01, to induce cell proliferation and arrest of differentiation. We analyzed colony formation efficiency, organoid size and morphology, cell proliferation and, finally, gene expression by RT-qPCR. Results: An average of 85 ± 7 million (n=6) EpCAM/LGR5 enriched fetal hBTSCs were obtained. The cells isolated from fetal biliary tree showed a high tendency to generate organoids with high colony formation efficiency (>90%). After 5 days in culture, the organoids were microscopically detected as spherical structures and after 7 days, they reached a macroscopically visible size. Cell proliferation in organoids was significantly higher compared to 2D conditions (p<0.05). Fetal biliary tree organoids were composed of single layered cuboidal epithelium and inner cell masses. RT-qPCR analysis indicated that organoids expressed multipotency stem cell markers (SOX2, NANOG, OCT4), endodermal stem/progenitor cell markers (LGR5, EpCAM, PDX1, SOX17), hepatic, pancreatic and ductal markers (ALB, CYPA3, INS, CFTR, CK19) and stem/progenitor surface genes (NCAM, CD133, CD44), recapitulating major processes of self-organization during embryonic development. Specifically, organoids expressed a higher level of LGR5 compared to 2D cultures (p<0.05).we have demonstrated expand organoids stably in vitro for at least two months when they remained phenotypically stable and suitable for regenerative medicine programs. Conclusion: We have demonstrated that organoids expand clonogenically stable in vitro for at least two months, maintaining a stable phenotype of multipotent stem cells. This system has potential applications in regenerative medicine of liver and pancreas and in disease modeling.