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June 06-07, 2019 | London, UK
2nd International Conference on
Tissue Science and Molecular Biology,
Stem Cells & Separation Techniques
Joint Event
Biomedical Research (An International Journal of Medical Sciences) | ISSN: 0976-1683 Volume 30
Generation of 3d organoids of human fetal biliary tree stem cells (hbtscs) as
innovative tool for the regenerative medicine of liver and pancreas
S Safarikia
Sapienza University of Rome, Italy
3D organoids represent an advanced culture technology
in the field of stem cells and regenerative medicine,
recapitulating embryonic organ development. 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 and differentiate them toward hepatocyte
cells which are suitable for cell therapy and regenerative
medicine of liver. The fetal biliary tree (N=3, obtained from
elective pregnancy termination) was digested, mechanically
and enzymatically, to isolate EpCAM/LGR5 -enriched hBTSCs,
we alsoused the fragments of undigestedbileduct to cultivate
the organoids. Cells and bile duct fragments were then
embedded in Matrigel and cultured in an expansion organoid
medium containing soluble factors typical of the stem cell
niche (e.g. EGF, FGF, Noggin, R-Spondin1) that 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 to induce cell proliferation and arrest
of differentiation. After 7 days the medium was changed to
differentiation medium for a period of 10 days. We analyzed
colony formation efficiency, organoid size and morphology,
cell proliferation and gene expression by RT-qPCR. An average
of 85 ± 7 million (N=3) 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 (> 60%). 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 and population doubling 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 demonstrated that organoids in expansion
condition expressed multipotency stem cell markers (SOX2,
NANOG, OCT4), endodermal stem/progenitor cell markers
(LGR5, EpCAM, PDX1, SOX17), hepatic progenitors and ductal
markers (CK19, CK7) and stem/progenitor surface genes
(NCAM, CD133, CD44), recapitulating major processes of
self-organization during embryonic development, whereas
the differentiated organoids expressed high level of mature
hepatocyte marker like CYP3A and ALB. Interestingly, LGR5
Expression reduced notably in organoids in differentiation
condition compared to expansion condition (p< 0.01).
Moreover, differentiated organoids acquired a hepatocyte
morphology, including polygonal cell shape and secreted
significant high level of albumin into medium respect to the
samecellsin2Dculture.Wehavedemonstratedthatorganoids
expand clonogenically stable in vitrofor at least two months,
maintaining a stable phenotype of multipotent stem cells and
they can differentiate toward mature functional hepatocyte.
This system has potential applications in regenerative
medicine of liver and pancreas and in disease modelling.
e:
Biomed Res, Volume 30
ISSN: 0976-1683